Composition of lipid-based nanoparticles for small molecules and macromolecules

ABSTRACT

Described herein are nanoparticles comprising a mixture of a steroid, a phospholipid composition, an α-tocopheryl compound, and a therapeutic agent wherein the α-tocopheryl compound is presented on the surface of the nanoparticle. In some embodiments, the nanoparticles are useful for delivering a peptide or a protein. In some embodiments, the nanoparticles are formulated for ocular administration. In other embodiments, the nanoparticles are formulated to cross the blood brain barrier for the delivery of the therapeutic agents to the brain.

The present application claims benefit of priority to U.S. ProvisionalApplication Ser. No. 62/258,030, filed Nov. 20, 2015, the entirecontents of which are hereby incorporated by reference.

The invention was made with government support under Grant No. R03NS087322-01 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND

1. Field of the Disclosure

The present disclosure relates generally to the fields of medicine andpharmaceutics. In particular, it relates to nanoparticle compositionswhich target scavenger receptor class B type I (SR-BI) and delivertherapeutic agents to locations which express this receptor such as thebrain, the eyes, and tumors.

2. Description of Related Art

Nerve Growth Factor (NGF) is one of the members of the neurotrophinfamily with multifaceted functions. It is well known for its role insurvival, maintenance and differentiating actions on sympathetic andsensory neurons of peripheral nervous system and for the maintenance offunctional integrity of cholinergic neurons in the central nervoussystem (CNS) (Aloe et al., 2012). Beneficial effects of NGF in variousdisease conditions, such as peripheral neuropathies, diabetes, skinulcers, human immune deficiency virus, and ophthalmology, make it apotential therapeutic protein (Sofroniew et al., 2001). However, NGFadministration through various routes like intravenous, subcutaneous orintra-cerebro ventricular (ICV) infusions caused a variety ofundesirable and unwanted effects in patients (Apfel, 2002; McArthur etal., 2000; Eriksdotter Jonhagen et al., 1998). CERE-110, which wasdiscontinued in Phase II clinical trial, is an adeno-associated viralvector that encodes the gene for NGF (Mandel, 2010). NsG0202 consists ofan implantable encapsulated cell biodelivery device that secretes NGF(Wahlberg et al., 2012). However, both CRE-110 and NsG0202 are invasiverequiring brain surgery procedures to incorporate them into the certainlocations of the brain.

The use of proteins in medicine has been limited by their poor stabilityto proteolytic and hydrolytic degradation, low permeability across thebarriers, and short biologic half-life in the circulatory system (PintoReis et al., 2006; Vaishya et al., 2015). Indeed, the half-life of NGFwas about 5.4 min by intravenous injection in adult rats (Tria et al.,1994). Nanoparticles (NPs) are promising delivery systems for NGF. Thus,NPs could offer improved transport properties and pharmacokineticprofiles after systemic administration. Moreover, NPs could enhancebiodistribution, modify release characteristics, reduce immunogenicityand target identified tissue with minimal distribution to normal tissues(Zhang et al., 2009). NPs have been studied to deliver NGF to cross theblood-brain barrier (BBB). NGF was absorbed on poly(butyl cyanoacrylate)(PBCA) NPs coated with polysorbate 80 (Kurakhmaeva et al., 2009). Theresults demonstrated that polysorbate-coated NGF PBCA NPs were able tocross the BBB and showed the anti-Parkinson effect in mice. Althoughabout 96% of NGF was adsorbed onto the PBCA NPs after coating withpolysorbate 80, the total NGF loading was low at only about 1.2 ng/mLleading to possible toxicity induced by the high dose of PBCA NPsneeded. In other studies of the PBCA NPs, the competition of serumprotein competed with polysorbate 80 as well as the rapid NP degradationin serum/plasma induced desorption of compounds adsorbed onto PBCA NPswithin a few minutes (Olivier, 2005). Additionally, NGF has beendirectly incorporated into a liposome delivery system coated with RMP-7to target to B2 receptor on brain microvascular endothelial cells.However, the entrapment efficiency (EE) of NGF was low at about 34% (Xieet al., 2005). NGF was also conjugated to an antibody of the transferrinreceptor (Granholm et al., 1994) and the composition was able crossedthe blood-brain barrier after peripheral injection. However, similarwith the PBCA NPs, NGF was not protected by the formulations fromdegradation by enzymes. All of these compositions have significantissues which prevent them from becoming potential commercialapplications for the delivery of NGF.

The studies showed that α-tocopherol originated from plasma isassociated with HDLs and transported by scavenger receptor class B, typeI (SR-BI) across the BBB (Balazs et al., 2004). Additional studies havedemonstrated that HDL-associated α-tocopherol was taken up in 10-foldexcess of HDL holoparticles, indicating efficiently selective uptakemediated by the SR-BI to cross the BBB (Goti et al., 2001).D-α-Tocopheryl polyethylene glycol succinate (vitamin E TPGS or simplyTPGS) is a water soluble source of vitamin E with extended half-life andenhanced cellular uptake of the drug due to the combination of PEG andvitamin E (Structures shown in FIG. 1). Other nanoparticle compositionscontaining TPGS and other vitamin E derivatives have been developed bythe inventors but these compositions do not present TPGS on the surfaceof the nanoparticle (Dong et al., 2009).

Similarly, delivery of therapeutic macromolecules, such as peptides,proteins, nucleic acids, polymers, or other large molecules, into theeye has been technically challenging. The anatomical structures of theeye limit entry of unaided foreign molecules into the intraocular space.As of now, other than intraocular injection, there is no clinicallypractical means to deliver therapeutically sufficient amount ofmacromolecules inside the eye. In the eye, SR-BI is also highlyexpressed at the corneal epithelial and endothelial cells, in thechoroidal and scleral cells (Provost, 2003). The expression of thisreceptor facilitates selective uptake of high-density lipoprotein(HDL)-associated cholesteryl ester and α-tocopherol by receptor-mediatedtransport in the eyes. In addition, SR-BI has been shown to beoverexpressed in many cancer cells. Therefore, the new compositions andmethods for the drug delivery in this invention provide a novel,nonobvious, and useful approach to overcome the difficulties in drugdelivery.

Therefore, new compositions and methods for the delivery of therapeuticagents which present α-tocopheryl compounds on the surface which areable to cross the blood brain barrier.

SUMMARY

In some aspects, the present disclosure provides compositions which areformulated to target the scavenger receptor class B type I (SR-BI). Insome embodiments, these compositions may be used to deliver therapeuticagents to any target which expresses the scavenger receptor class B typeI including the brain, to a tumor, or to the eyes. In some embodiments,other targeted ligands may be coated on the surface of nanoparticle totarget other receptors.

In some aspects, the present disclosure provides compositionscomprising:

-   -   (a) a therapeutic agent;    -   (b) an α-tocopheryl compound;    -   (c) a phospholipid composition; and    -   (d) a steroid or steroid derivative,        wherein the composition is formulated as a nanoparticle and the        α-tocopheryl compound is substantially located on the surface of        the nanoparticle.

In other aspects, the present disclosure provides compositionscomprising:

-   -   (a) a therapeutic agent;    -   (b) an α-tocopheryl compound;    -   (c) a phospholipid composition;    -   (d) a steroid or steroid derivative, and    -   (e) an apolipoprotein;        wherein the composition is formulated as a nanoparticle and the        α-tocopheryl compound is substantially located on the surface of        the nanoparticle.

In some embodiments, the therapeutic agent is a therapeutic protein. Insome embodiments, the therapeutic protein is a growth factor. In someembodiments, the therapeutic protein is a neurotrophic factor. In otherembodiments, the therapeutic protein is a neurotrophin such as nervegrowth factor. In other embodiments, the neurotrophin is brain-derivedneurotrophic factor, neurotrophin-3, or neurotrophin-4. In someembodiments, the neurotrophic factor is brain-derived neurotrophicfactor, ciliary-derived neurotrophic factor, basic fibroblast growthfactor, nerve growth factor, glial cell line-derived neurotrophicfactor, neurotrophin-3, or neurotrophin-4. In some embodiments, theneurotrophic factor is brain-derived neurotrophic factor,ciliary-derived neurotrophic factor, or basic fibroblast growth factor.

In other embodiments, the therapeutic protein is an antibody. In otherembodiments, the therapeutic protein is a mixture of antibodies. In someembodiments, the antibody is an anti-vascular endothelial growth factor(VEGF) antibody. In some embodiments, the antibody mixture reduces thebiological activity of VEGF. In other embodiments, the therapeuticprotein is a protein which binds VEGF. In some embodiments, thetherapeutic protein is a mixture of proteins which binds VEGF. In someembodiments, the protein which binds VEGF reduces the biologicalactivity of VEGF. In some embodiments, the proteins which bind VEGFreduce the biological activity of VEGF. In some embodiments, the proteinbinds placental growth factor (PIGF) and reduces the biological activityof PIGF. In some embodiments, the proteins bind PIGF and reduce thebiological activity of PIGF. In some embodiments, the protein binds VEGFand PIGF and reduces the biological activity of both factors. In someembodiments, the proteins bind VEGF and PIGF and reduce the biologicalactivity of both factors.

In some embodiments, the therapeutic protein is a mixture of atherapeutic protein and a polycationic protein molecule. In someembodiments, the polycationic protein molecule is polylysine,polyarginine, or protamine. In some embodiments, the polycationicprotein molecule is protamine. In some embodiments, the therapeuticagent is a chemotherapeutic compound. In some embodiments, thetherapeutic agent is a taxane such as docetaxel. In some embodiments,the therapeutic agent is a composition comprising a chemotherapeuticagent and a therapeutic oligonucleotide. In some embodiments, thetherapeutic oligonucleotide is an antisense oligonucleotide. In someembodiments, the therapeutic oligonucleotide is an antisenseoligonucleotide which reduces the expression of secretory clusterin(sCLU). In some embodiments, the therapeutic oligonucleotide is OGX-011.

In some embodiments, the α-tocopheryl compound is a pegylated derivativeof α-tocopheryl. In some embodiments, the pegylated derivative ofα-tocopheryl facilitates transportation of the composition across theblood-brain barrier. In some embodiments, the α-tocopheryl compoundcomprises a polyethylene glycol group with a molecular weight from about100 g/mol to about 5000 g/mol. In some embodiments, the polyethyleneglycol group has a molecular weight from about 500 g/mol to about 2500g/mol. In some embodiments, the polyethylene glycol group has amolecular weight of about 1000 g/mol. In some embodiments, thepolyethylene glycol group is linked to the α-tocopheryl compound by alinker group. In some embodiments, the linker group is a succinategroup. In some embodiments, the α-tocopheryl compound is d-α-tocopherylpolyethylene glycol 1000 succinate.

In some embodiments, the phospholipid composition comprises two or morephospholipids. In some embodiments, the phospholipid compositioncomprises 2, 3, 4, 5, 6, 7, or 8 phospholipids. In some embodiments, thephospholipid composition comprises a mixture of phospholipids,triglycerides and apolipoprotein A-I which mimic a high densitylipoprotein. In sonic embodiments, the phospholipid compositioncomprises a mixture of phospholipids and triglycerides. In someembodiments, the phospholipid composition comprises a first phospholipidof the formula:

wherein:

-   -   R₁ and R₂ are each independently alkyl_((C6-24)),        alkenyl_((C6-24)), or a substituted version of either of these        groups; and    -   R₃ is hydrogen or —(CH₂)_(x)R_(a), wherein:        -   x is 1, 2, 3, 4, 5, or 6; and        -   R_(a) is —NR′R″R′″⁺ or —CH(CO₂R_(b))NR_(c)R_(d), wherein:            -   R′, R″ and R′″ are each independently hydrogen,                alkyl_((C≦6)), or substituted alkyl_((C≦6)); and            -   R_(b), R_(c), and R_(d) are each independently hydrogen,                alkyl_((C≦6)), or substituted alkyl_((C≦6));                or a salt thereof.

In some embodiments, R₁ is alkyl_((C6-24)). In other embodiments, R₁ isalkenyl_((C6-24)). In some embodiments, R₂ is alkyl_((C6-24)). In otherembodiments, R₂ is alkenyl_((C6-24)). In some embodiments, R₃ is—(CH₂)_(x)R_(a), wherein:

-   -   x is 1, 2, 3, 4, 5, or 6; and    -   R_(a) is —NR′R″R′″+, wherein: R′, R″, and R′″ are each        independently hydrogen, alkyl_((C≦6)), or substituted        alkyl_((C6≦6)).

In some embodiments, x is 1, 2, 3, or 4. In some embodiments, x is 1, 2,or 3. In some embodiments, x is 1. In other embodiments, x is 2. Inother embodiments, x is 3. In some embodiments, R_(a) is —NH₃ ⁺. Inother embodiments, R_(a) is —N(CH₃)₃ ⁺. In some embodiments, R_(a) is—CH(CO₂R_(b))NR_(c)R_(d), wherein:

-   -   R_(b), R_(c), and R_(d) are each independently hydrogen,        alkyl_((C≦6)), or substituted alkyl_((C≦6)),

In some embodiments, R_(b) is hydrogen. In some embodiments, R_(c) ishydrogen. In some embodiments, R_(d) is hydrogen. In some embodiments,R₃ is hydrogen. In some embodiments, the first phospholipid is furtherdefined by the structure:

In other embodiments, the first phospholipid is further defined by thestructure:

In some embodiments, the first phospholipid is a phosphatidylcholine.

In some embodiments, the phospholipid composition further comprises asecond phospholipid. In some embodiments, the second phospholipid isfurther defined by formula I and wherein the second phospholipid isdifferent from the first phospholipid. In some embodiments, the secondphospholipid is further defined as:

In other embodiments, the second phospholipid is further defined as:

In some embodiments, R₁ is alkyl_((C6-24)). In other embodiments, R₁ isalkenyl_((C6-24)). In some embodiments, R₂ is alkyl_((C6-24)). In otherembodiments, R₂ is alkenyl_((C6-24)). In some embodiments, the secondphospholipid is a phosphatidylserine compound.

In some embodiments, the phospholipid composition further comprises athird phospholipid compound. In some embodiments, the third phospholipidcompound is a compound of the formula:

wherein:

-   -   R₄ and R₅ are each independently alkyl_((C6-24)),        alkenyl_((C6-24)), or a substituted version of either of these        groups; and    -   R₆ is hydrogen or —(CH₂)_(x)R_(a), wherein:        -   x is 1, 2, 3, 4, 5, or 6, and        -   R_(a) is —NR′R″R′″⁺ or —CH(CO₂R_(b))NR_(c)R_(d), wherein:            -   R′, R″, and R′″ are each independently hydrogen,                alkyl_((C≦6)), or substituted alkyl_((C≦6)); and            -   R_(b), R_(c), and R_(d) are each independently hydrogen,                alkyl_((C≦6)), or substituted alkyl_((C≦6));    -   R₇ is hydroxy or alkoxy_((C≦6)), acyloxy_((C≦6)), or a        substituted version of either of these groups;        or a salt thereof.

In some embodiments, R₄ is alkyl_((C6-24)). In other embodiments, R₄ isalkenyl_((C6-24)). In some embodiments, R₅ is alkyl_((C6-24)). In otherembodiments, R₅ is alkenyl_((C6-24)). In some embodiments, R₆ is—(CH₂)_(x)R_(a), wherein:

-   -   x is 1, 2, 3, 4, 5, or 6; and    -   R_(a) is —NR′R″R′″⁺, wherein: R′, R″, and R′″ are each        independently hydrogen, alkyl_((C≦6)), or substituted        alkyl_((C≦6)).

In some embodiments, x is 1, 2, 3, or 4. In some embodiments, x is 1, 2,or 3. In some embodiments, x is 1. In other embodiments, x is 2. Inother embodiments, x is 3. In some embodiments, R_(a) is —NH₃ ⁺. Inother embodiments, R_(a) is —N(CH₃)₃ ⁺. In some embodiments, R₇ ishydroxy. In other embodiments, R₇ is alkoxy_((C≦6)). In otherembodiments, R₇ is acyloxy_((C≦6)). In some embodiments, the thirdphospholipid compound is further defined by the formula:

In some embodiments, the steroid or steroid derivative is a cholesterolester_((C≦24)). In some embodiments, the steroid or steroid derivativeis a cholesterol. In other embodiments, the steroid or steroidderivative is cholesterol oleate.

In some embodiments, the compositions further comprise an apoliprotein.In some embodiments, the apoliprotein is apolipoprotein A1. In otherembodiments, the apoliprotein is a modified apolipoprotein A1. In someembodiments, the compositions further comprise a cell permeablizingagent. In some embodiments, the cell permeabilizing agent is apolyarginine peptide. In other embodiments, the cell permeabilzing agentis a pegylated polyarginine. In some embodiments, the compositionsfurther comprise a targeting agent. In some embodiments, the targetingagent is an antibody, an antibody fragment, a peptide, a protein, anucleic acid, or a small molecule. In some embodiments, the compositionmay further comprise an endosomal escaping agent, such as MGDG,diacylglycerol, a polyphosphoinositide or a fatty acid.

In some embodiments, the ratio of the phospholipid composition to thesteroid or steroid derivative is from about 1:5 to about 15:1. In someembodiments, the ratio is 1:1 to about 10:1. In some embodiments, theratio is from about 4:1 to about 8:1. In some embodiments, the ratio isabout 4.9:1. In some embodiments, the ratio of the phospholipids in thephospholipid composition comprises a phosphatidylcholine tosphingomyelin ratio from about 10:1 to about 1:2. In some embodiments,the ratio is about 8:1 to about 2:1. In some embodiments, the ratio isabout 5.2:1. In some embodiments, the ratio of the phospholipids in thephospholipid composition comprises a phosphatidylcholine tophosphotidylserine ratio from about 25:1 to about 1:1. In someembodiments, the ratio is from about 20:1 to about 10:1. In someembodiments, the ratio is about 15.7:1. In some embodiments, the steroidor steroid derivative comprises 0.5 w/w % to about 12.5 w/w % of thecomposition. In some embodiments, the steroid or steroid derivativecomprises from about 2 w/w % to about 8 w/w %. In some embodiments, thesteroid or steroid derivative comprises about 4.8 w/w %. In someembodiments, the phospholipid composition comprises from about 10 w/w %to about 45 w/w % of the composition. In some embodiments, thephospholipid composition comprises 15 w/w % to about 30 w/w %. In someembodiments, the phospholipid composition comprises about 23.6 w/w %. Insome embodiments, the α-tocopheryl compound comprises from about 5 w/w %to about 60 w/w % of the composition. In some embodiments, theα-tocopheryl compound comprises from about 10 w/w % to about 50 w/w %.In some embodiments, the α-tocopheryl compound comprises about 14.8 w/w%.

In some embodiments, the therapeutic agent comprises from about 0.5 w/w% to about 25 w/w %. In some embodiments, the therapeutic agentcomprises from about 1.0 w/w % to about 15 w/w %. In some embodiments,the therapeutic agent comprises about 3.2 w/w %. In other embodiments,the therapeutic agent comprises about 10 w/w %. In some embodiments, thecompositions comprise the therapeutic agent and a polycationic moleculein a ratio from about 10:1 to about 1:10. In some embodiments, the ratiois from about 2:1 to about 1:2. In some embodiments, the ratio is about1:1. In some embodiments, the lipoprotein comprises from about 20 w/w %to about 70 w/w % of the composition. In some embodiments, thelipoprotein comprises from about 40 w/w % to about 60 w/w %. In someembodiments, the lipoprotein comprises about 50.8 w/w %.

In some embodiments, the nanoparticle has a particle size from about 100nm to about 500 nm. In some embodiments, the particle size is from about100 nm to about 200 nm. In some embodiments, the particle size is fromabout 130 nm to about 170 nm. In other embodiments, the particle size isfrom about 200 nm to about 300 nm. In some embodiments, the particlesize is from about 220 nm to about 270 nm. In some embodiments, thepolydispersity index is less than 0.3. In some embodiments, thepolydispersity index is less than 0.28.

In some embodiments, the compositions further comprise apharmaceutically acceptable carrier. In some embodiments, thecomposition is formulated for administration: ocularly, oculartopically, intracamerally, subretinally, peribulbarly, retrobulbarly,orally, intraadiposaliy, intraarterially, intraarticularly,intracranially, intradermally, intralesionally, intramuscularly,intranasally, intraocularly, intrapericardially, intraperitoneally,intrapleurally, intraprostatically, intrarectally, intrathecally,intratracheally, intratumorally, intraumbilically, intravaginally,intravenously, intravesicularly, intravitreally, liposomally, locally,mucosally, parenterally, rectally, subconjunctivally, subchoroidally,subcutaneously, sublingually, topically, transbuccally, transdermally,vaginally, in cremes, in lipid compositions, via a catheter, via alavage, via continuous infusion, via infusion, via inhalation, viainjection, via local delivery, or via localized perfusion. In someembodiments, the compositions are formulated for administration viaocular topical administration, eye drop, or as an injection. In someembodiments, the compositions are formulated for ocular administration.

In another aspects, the present disclosure provides methods of preparinga therapeutic agent-loaded nanoparticle comprising:

-   -   (a) admixing a composition with an organic solvent and        cholesterol, a composition with an organic solvent and a        phospholipid composition, a composition with an organic solvent        and an α-tocopheryl compound, and a composition with a solvent        and a therapeutic agent to form a first reaction mixture;    -   (b) removing the organic solvent from the first reaction mixture        to form a second reaction mixture;    -   (c) admixing the second reaction mixture to water by using a        homogenizer or a sonication probe to form a prototype        nanoparticle; and    -   (d) admixing one or more therapeutic agents with the prototype        nanoparticle to form a therapeutic agent-loaded nanoparticle.

In yet another aspect, the present disclosure provides methods ofpreparing a therapeutic agent-loaded HDL mimicking nanoparticlecomprising:

-   -   (a) admixing a composition with an organic solvent and        cholesterol, a composition with an organic solvent and a        phospholipid composition, and a composition with an organic        solvent and an α-tocopheryl compound to form a first reaction        mixture;    -   (b) removing the organic solvent from the first reaction mixture        to form a second reaction mixture;    -   (c) admixing the second reaction mixture to water to form a        prototype nanoparticle.    -   (d) admixing one or more therapeutic agents with the prototype        nanoparticle to form a therapeutic agent-loaded nanoparticle;        and    -   (e) admixing the therapeutic agent-loaded nanoparticle with        apolipoprotein A-I to form a therapeutic agent-loaded        HDL-mimicking nanoparticle.

In some embodiments, the methods further comprise homogenizing theprototype nanoparticle. In other embodiments, the methods furthercomprise homogenizing the therapeutic agent-loaded nanoparticle. In someembodiments, the organic solvent has a boiling point of less than 100°C. In some embodiments, the organic solvent is an alcohol_((C≦8)) suchas ethanol.

In some embodiments, the methods further comprise admixing one or moretherapeutic agents, wherein admixing one or more therapeutic agentscomprises:

-   -   (a) adding the therapeutic agent to the prototype nanoparticle;    -   (b) incubating the prototype nanoparticle and the therapeutic        agent for a first time period at a first temperature; and    -   (c) stirring the prototype nanoparticle and the therapeutic        agent for a second time period at a second temperature to form a        therapeutic agent-loaded nanoparticle.

In some embodiments, the first time period is from about 1 minute toabout 4 hours. In some embodiments, the first time period is from about5 minutes to about 2 hours. In some embodiments, the first time periodis about 30 minutes. In some embodiments, the first temperature is fromabout 25° C. to about 75° C. In some embodiments, the first temperatureis from about 30° C. to about 50° C. In some embodiments, the firsttemperature is about 37° C.

In some embodiments, the second time period is from about 1 minute toabout 4 hours. In some embodiments, the second time period is from about5 minutes to about 2 hours. In some embodiments, the second time periodis about 30 minutes. In some embodiments, the second temperature is fromabout 0° C. to about 37° C. In some embodiments, the second temperatureis from about 15° C. to about 37° C. In some embodiments, the secondtemperature is about 25° C.

In some embodiments, the methods further comprise admixing a protein orpeptide to the therapeutic agent-loaded HDL mimicking nanoparticle toform a protein and therapeutic agent-loaded HDL mimicking nanoparticle.In some embodiments, admixing the targeting agent comprises:

-   -   (a) adding the protein or peptide; and    -   (b) stirring the protein or peptide and the HDL mimicking        nanoparticle for a third time period at a third temperature to        obtain a protein and therapeutic agent-loaded HDL mimicking        nanoparticle.

In some embodiments, the protein or peptide is an apolipoprotein. Insome embodiments, the apolipoprotein is apolipoprotein A1. In someembodiments, the protein or peptide is a cell permabilizing agent. Insome embodiments, the targeting agent is a R11 peptide comprising a PEGgroup.

In some embodiments, the third time period is from about 2 hours toabout 24 hours. In some embodiments, the third time period is from about6 hours to about 18 hours. In some embodiments, the third time period isabout 12 hours. In some embodiments, the third temperature is from about0° C. to about 37° C. In some embodiments, the third temperature is fromabout 15° C. to about 37° C. In some embodiments, the third temperatureis about 25° C. In some embodiments, the homogenization compriseshomogenizing the nanoparticle for a fourth time period from about 10seconds to about 30 minutes. In some embodiments, the fourth time periodis from about 30 seconds to about 10 minutes. In some embodiments, thefourth time period is about 5 minutes.

In still another aspect, the present disclosure provides compositionsprepared according to the methods described herein.

In yet another aspect, the present disclosure provides methods oftreating a disease or disorder in a patient comprising administering tothe patient a therapeutically effective amount of a compositiondescribed herein.

In some embodiments, the disease or disorder is a central nervous systemdisorder. In some embodiments, the central nervous system disorder isAlzheimer's disease, Parkinson's disease, stroke, dementia, depression,schizophrenia, autism, Rett syndrome, anorexia nervosa, and bulimianervosa. In other embodiments, the disease is an inflammatory disease.In some embodiments, the inflammatory disease is a rheumatic disease andmultiple sclerosis. In other embodiments, the disease is acardiovascular disease. In some embodiments, the cardiovascular diseaseis atherosclerosis, obesity, type 2 diabetes and metabolic syndrome. Inother embodiments, the disease or disorder is cancer. In someembodiments, the cancer is a carcinoma, sarcoma, lymphoma, leukemia,melanoma, mesothelioma, multiple myeloma, or seminoma. In someembodiments, the cancer is of the bladder, blood, bone, brain, breast,central nervous system, cervix, colon, endometrium, esophagus, gallbladder, gastrointestinal tract, genitalia, genitourinary tract, head,kidney, larynx, liver, lung, muscle tissue, neck, oral or nasal mucosa,ovary, pancreas, prostate, skin, spleen, small intestine, largeintestine, stomach, testicle, or thyroid. In some embodiments, thecancer is prostate cancer such as a metastatic castration resistantprostate cancer. In other embodiments, the cancer is brain cancer.

In some embodiments, the methods further comprise administering a secondanti-cancer therapy. In some embodiments, the second anti-cancer therapyis a second chemotherapeutic compound, radiotherapy, immunotherapy, orsurgery.

In other embodiments, the disease or disorder is a disease or disorderof the eye. In some embodiments, the disease or disorder of the eye isglaucoma, age-related macular degeneration, diabetic retinopathy,retinal ischemic abnormalities, uveitis, endophthalmitis, or optic nervetrauma. In some embodiments, the disease or disorder is glaucoma.

In some embodiments, the patient is a mammal. In some embodiments, thepatient is a human. In some embodiments, the composition is administeredonce. In other embodiments, the composition is administered two or moretimes.

In yet another aspect, the present disclosure provides methods ofinducing neuronal growth comprising administering a compositiondescribed herein. In some embodiments, the composition is administeredin vitro. In other embodiments, the composition is administered in vivo.In some embodiments, the composition is administered to a neuron.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein.

The terms “comprise” (and any form of comprise, such as “comprises” and“comprising”), “have” (and any form of have, such as “has” and“having”), “contain” (and any form of contain, such as “contains” and“containing”), and “include” (and any form of include, such as“includes” and “including”) are open-ended linking verbs. As a result, amethod, composition, kit, or system that “comprises,” “has,” “contains,”or “includes” one or more recited steps or elements possesses thoserecited steps or elements, but is not limited to possessing only thosesteps or elements; it may possess (i.e., cover) elements or steps thatare not recited. Likewise, an element of a method, composition, kit, orsystem that “comprises,” “has,” “contains,” or “includes” one or morerecited features possesses those features, but is not limited topossessing only those features; it may possess features that are notrecited.

Any embodiment of any of the present methods, composition, kit, andsystems may consist of or consist essentially of—rather thancomprise/include/contain/have—the described steps and/or features. Thus,in any of the claims; the term “consisting of” or “consistingessentially of” may be substituted for any of the open-ended linkingverbs recited above, in order to change the scope of a given claim fromwhat it would otherwise be using the open-ended linking verb.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

Following long-standing patent law, the words “a” and “an,” when used inconjunction with the word “comprising” in the claims or specification,denotes one or more, unless specifically noted.

As used in this application, the term “average molecular weight” refersto the relationship between the number of moles of each polymer speciesand the molar mass of that species. In particular, each polymer moleculemay have different levels of polymerization and thus a different molarmass. The average molecular weight can be used to represent themolecular weight of a plurality of polymer molecules. Average molecularweight is typically synonymous with average molar mass. In particular,there are three major types of average molecular weight: number averagemolar mass, weight (mass) average molar mass, and Z-average molar mass.In the context of this application, unless otherwise specified, theaverage molecular weight represents either the number average molar massor weight average molar mass of the formula. In some embodiments, theaverage molecular weight is the number average molar mass. In someembodiments, the average molecular weight may be used to describe a PEGcomponent present as a part of the α-tocopheryl compound.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating certain embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1—The structure comparison of vitamin E TPGS and α-tocopheryl.

FIG. 2—Influence of homogenization times on NP preparation. Excipientswere homogenized for 0, 1, 2, 3, 4, 5 or 6 min to form the NPs. Data arepresented as the mean of particle size ±SD (n=3). #p>0.05.

FIG. 3—Relationship of Apo A-I loading and EE %. #p>0.05 for the EE %.

FIG. 4—Separation of the NGF NPs from free NGF by a gel filtrationSeparhose CL-4B column. The NGF NPs were measured based on particleintensity. Free NGF was measured by a Sandwich ELISA method.

FIG. 5—Long-term stability of batch 4-2 that did not contain Apo A-I.The batch was monitored for particle size and P.I. over 6 months. Datapresented as mean particle size.

FIG. 6—Long-term stability of the prototype HDL-mimickingα-tocopheryl-coated NPs. Batch 2-4, 2-6, and 2-7 in Table 2B consist ofthree different compositions. Each batch was prepared in triplicate andmonitored for particle size and P.I. over three months. For all testedNPs, P.I.<0.3. Data are presented as the mean particle size of threebatches at the certain composition. #p>0.05. within the group.

FIG. 7—DTX NPs decreased IC₅₀ in DTX-resistant DU145 cells at 72 h (*p<0.05).

FIG. 8. Uptake of cy5-labeled miRNA363 NPs in prostate cancer cells by aconfocal microscopy. The pictures show the imaging at the centralsection of cells analyzed by Z-stack image.

FIG. 9. Intercellular uptake of FITC-siRNA NPs in NCI/ADR-RES cells. Thecells were treated with free FITC-siRNA and FITC-siRNA NPs for 3 hours.The Z-stack imaging was detected by a confocal microscope.

FIG. 10. Cellular uptake of Cy3-labeled anti-GAPDH siRNA NPs on PC3cells (prostate cancer cells). The PC3 cells were treated with freesiRNA or siRNA-loaded NPs for 4 hours at 37° C., which had an equivalentconcentration of siRNA (6.20 μg/mL); The Z-stack imaging was taken byusing a confocal microscope.

FIG. 11. Structure of MGDG (monogalactosyldiacyldiacylglycerol), anonionic and non-bilayer lipid.

FIG. 12. Luciferase knockdown of anti-luciferase siRNA nanoparticles. Inall of the treatments, the concentration of anti-luciferase siRNA was12.3 pmole. Lipofectamine, a well-known commercial gene transfectionagent, was used as a positive control. DOPE was mixed with PC to formnanoparticles for comparison with MGDG NPs. MGDG was incorporated withTPGS or PC in different concentrations to form different nanoparticles,so that the inventors were able to treat cells with differentconcentrations of MGDG (5 μm, 25 μm and 50 μm). (* p<0.05: significantdifference compared to the control; #p>0.05: no significant differencecompared to lipofectamine according to t-test),

FIGS. 13A-B. imaging of neurite outgrowth when the cells were treatedwith 50 ng/ml of free NGF (FIG. 13A) and NGF HDL-mimicking NPs (FIG.13B).

FIG. 14. In vitro release profiles of free NGF and NGF NPs in 5% BSA-PBSsolution (pH 7). Data are presented as the mean SD (n=4).

FIG. 15. Biodistribution of NGF NPs after mice were intravenouslyinjected 40 μg/kg of NGF for 30 min (n=3). NGF NPs resulted insignificantly higher NGF concentration in plasma compared to free NGF(p<0.05). For other tissues, NGF NPs led to lower NGF concentrationscompared to free NGF (p<0.05).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In some aspects, the present disclosure provides nanoparticles which maybe used to deliver therapeutic agents using nanoparticles which arecoated with α-tocopheryl compounds. These compounds may be used todelivery compounds by crossing the blood retina barrier, blood brainbarrier, to cancer cells, or to other tissues or cells that expressscavenger receptor class B type I (SR-BI). In some embodiments, thesenanoparticles present on their surfaces compounds or components whichare recognized by scavenger receptor class B type I (SR-BI). Thesenanoparticles may be used with small molecule therapeutic agents,antibodies or functionalized antibodies, peptides, proteins, nucleicacids or functionalized nucleic acids, or other large moleculetherapeutic agents. In some embodiments, these nanoparticles may be usedto treat neurological disorders or ocular disorders.

I. Chemical Definitions

When used in the context of a chemical group: “hydrogen” means —H;“hydroxy” or “hydroxyl” means —OH; “oxo” means ═O; “carbonyl” means—C(═O)—; “carboxy” means —C(═O)OH (also written as —COOH or —CO₂H);“halo” means independently —F, —Cl, —Br or —I; “amino” means —NH₂;“hydroxyamino” means —NHOH; “nitro” means —NO₂; imino means ═NH; “cyano”means —CN; “isocyanate” means —N═C═O; “azido” means —N₃; in a monovalentcontext “phosphate” means —OP(O)(OH)₂ or a deprotonated form thereof; ina divalent context “phosphate” means —OP(O)(OH)O— or a deprotonated formthereof; “mercapto” means —SH; and “thio” means ═S; “sulfonyl” means—S(O)₂—; and “sulfinyl” means —S(O)—.

In the context of chemical formulas, the symbol “—” means a single bond,“═” means a double bond, and “≡” means triple bond. The symbol “ - - - -” represents an optional bond, which if present is either single ordouble. The symbol “

” represents a single bond or a double bond. Thus, for example, theformula

includes

And it is understood that no one such ring atom forms part of more thanone double bond. Furthermore, it is noted that the covalent bond symbol“—”, when connecting one or two stereogenic atoms, does not indicate anypreferred stereochemistry. Instead, it cover all stereoisomers as wellas mixtures thereof. The symbol “

”, when drawn perpendicularly across a bond (e.g.,

for methyl) indicates a point of attachment of the group. It is notedthat the point of attachment is typically only identified in this mannerfor larger groups in order to assist the reader in unambiguouslyidentifying a point of attachment. The symbol “

” means a single bond where the group attached to the thick end of thewedge is “out of the page.” The symbol “

” means a single bond where the group attached to the thick end of thewedge is “into the page”. The symbol “

” means a single bond where the geometry around a double bond (e.g.,either E or Z) is undefined. Both options, as well as combinationsthereof are therefore intended. Any undefined valency on an atom of astructure shown in this application implicitly represents a hydrogenatom bonded to that atom. A bold dot on a carbon atom indicates that thehydrogen attached to that carbon is oriented out of the plane of thepaper.

When a group “R” is depicted as a “floating group” on a ring system, forexample, in the formula:

then R may replace any hydrogen atom attached to any of the ring atoms,including a depicted, implied, or expressly defined hydrogen, so long asa stable structure is formed. When a group “R” is depicted as a“floating group” on a fused ring system, as for example in the formula:

then R may replace any hydrogen atom attached to any of the ring atomsof either of the fused rings unless specified otherwise. Replaceablehydrogen atoms include depicted hydrogen atoms (e.g., the hydrogen atomattached to the nitrogen in the formula above), implied hydrogen atoms(e.g., a hydrogen atom of the formula above that is not shown butunderstood to be present), expressly defined hydrogen atoms, andoptional hydrogen atoms whose presence depends on the identity of a ringatom (e.g., a hydrogen atom attached to group X, when X equals —CH—), solong as a stable structure is formed. In the example depicted, R mayreside on either the 5-membered or the 6-membered ring of the fused ringsystem. In the formula above, the subscript letter “y” immediatelyfollowing the group “R” enclosed in parentheses, represents a numericvariable. Unless specified otherwise, this variable can be 0, 1, 2, orany integer greater than 2, only limited by the maximum number ofreplaceable hydrogen atoms of the ring or ring system.

For the groups and classes below, the number of carbon atoms in thegroup is as indicated as follows: “Cn” defines the exact number (n) ofcarbon atoms in the group/class. “C≦n” defines the maximum number (n) ofcarbon atoms that can be in the group/class, with the minimum number assmall as possible for the group in question, e.g., it is understood thatthe minimum number of carbon atoms in the group “alkenyl_((C≦8))” or theclass “alkene_((C≦8))” is two. Compare with “alkoxy_((C≦10))”, whichdesignates alkoxy groups having from 1 to 10 carbon atoms. Also compare“phosphine_((C≦10))”, which designates phosphine groups having from 0 to10 carbon atoms. “Cn−n′” defines both the minimum (n) and maximum number(n′) of carbon atoms in the group. Thus, “alkyl_((C2-10))” designatesthose alkyl groups having from 2 to 10 carbon atoms. Typically thecarbon number indicator follows the group it modifies, is enclosed withparentheses, and is written entirely in subscript; however, theindicator may also precede the group, or be written without parentheses,without signifying any change in meaning. Thus, the terms “C5 olefin”,“C5-olefin”, “olefin_((C5))”, and “olefin_(C5)” are all synonymous.

The term “saturated” as used herein means the compound or group somodified has no carbon-carbon double and no carbon-carbon triple bonds,except as noted below. In the case of substituted versions of saturatedgroups, one or more carbon oxygen double bond or a carbon nitrogendouble bond may be present. And when such a bond is present, thencarbon-carbon double bonds that may occur as part of keto-enoltautomerism or imine/enamine tautomerism are not precluded.

The term “aliphatic” when used without the “substituted” modifiersignifies that the compound/group so modified is an acyclic or cyclic,but non-aromatic hydrocarbon compound or group. In aliphaticcompounds/groups, the carbon atoms can be joined together in straightchains, branched chains, or non-aromatic rings (alicyclic). Aliphaticcompounds/groups can be saturated, that is joined by single bonds(alkanes/alkyl), or unsaturated, with one or more double bonds(alkenes/alkenyl) or with one or more triple bonds (alkynes/alkynyl).

The term “alkyl” when used without the “substituted” modifier refers toa monovalent saturated aliphatic group with a carbon atom as the pointof attachment, a linear or branched acyclic structure, and no atomsother than carbon and hydrogen. The groups —CH₃ (Me), —CH₂CH₃ (Et),—CH₂CH₂CH₃ (n-Pr or propyl), —CH(CH₃)₂ (i-Pr, ^(i)Pr or isopropyl),—CH₂CH₂CH₂CH₃ (n-Bu), —CH(CH₃)CH₂CH₃ (sec-butyl), —CH₂CH(CH₃)₂(isobutyl), —C(CH₃)₃ (tert-butyl, t-butyl, t-Bu or ^(t)Bu), and—CH₂C(CH₃)₃ (neo-pentyl) are non-limiting examples of alkyl groups. Theterm “alkanediyl” when used without the “substituted” modifier refers toa divalent saturated aliphatic group, with one or two saturated carbonatom(s) as the point(s) of attachment, a linear or branched acyclicstructure, no carbon-carbon double or triple bonds, and no atoms otherthan carbon and hydrogen. The groups —CH₂—(methylene), —CH₂CH₂—,—CH₂C(CH₃)₂CH₂—, and —CH₂CH₂CH₂— are non-limiting examples of alkanediylgroups. The term “alkylidene” when used without the “substituted”modifier refers to the divalent group ═CRR′ in which R and R′ areindependently hydrogen or alkyl. Non-limiting examples of alkylidenegroups include: ═CH₂, ═CH(CH₂CH₃), and ═C(CH₃)₂. An “alkane” refers tothe compound H—R, wherein R is alkyl as this term is defined above. Whenany of these terms is used with the “substituted” modifier one or morehydrogen atom has been independently replaced by —OH, —F, —Cl, —Br, —I,—NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃,—NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂. The following groups are non-limiting examplesof substituted alkyl groups: —CH₂OH, —CH₂Cl, —CF₃, —CH₂CN, —CH₂C(O)OH,—CH₂C(O)OCH₃, —CH₂C(O)NH₂, —CH₂C(O)CH₃, —CH₂OCH₃, —CH₂OC(O)CH₃, —CH₂NH₂,—CH₂N(CH₃)₂, and —CH₂CH₂Cl. The term “haloalkyl” is a subset ofsubstituted alkyl, in which the hydrogen atom replacement is limited tohalo (i.e. —F, —Cl, —Br, or —I) such that no other atoms aside fromcarbon, hydrogen and halogen are present. The group, —CH₂Cl is anon-limiting example of a haloalkyl. The term “fluoroalkyl” is a subsetof substituted alkyl, in which the hydrogen atom replacement is limitedto fluoro such that no other atoms aside from carbon, hydrogen andfluorine are present. The groups —CH₂F, —CF₃, and —CH₂CF₃ arenon-limiting examples of fluoroalkyl groups.

The term “alkenyl” when used without the “substituted” modifier refersto an monovalent unsaturated aliphatic group with a carbon atom as thepoint of attachment, a linear or branched acyclic structure, at leastone nonaromatic carbon-carbon double bond, no carbon-carbon triplebonds, and no atoms other than carbon and hydrogen. Non-limitingexamples include: —CH═CH₂ (vinyl), —CH═CHCH₃, —CH═CHCH₂CH₃, —CH₂CH═CH₂(allyl), —CH₂CH═CHCH₃, and —CH═CHCH═CH₂. The term “alkenediyl” when usedwithout the “substituted” modifier refers to a divalent unsaturatedaliphatic group, with two carbon atoms as points of attachment, a linearor branched, a linear or branched acyclic structure, at least onenonaromatic carbon-carbon double bond, no carbon-carbon triple bonds,and no atoms other than carbon and hydrogen. The groups —CH═CH—,—CH═C(CH₃)CH₂—, —CH═CHCH₂—, and —CH₂CH═CHCH₂— are non-limiting examplesof alkenediyl groups. It is noted that while the alkenediyl group isaliphatic, once connected at both ends, this group is not precluded fromforming part of an aromatic structure. The terms “alkene” or “olefin”are synonymous and refer to a compound having the formula H—R, wherein Ris alkenyl as this term is defined above. A “terminal alkene” refers toan alkene having just one carbon-carbon double bond, wherein that bondforms a vinyl group at one end of the molecule. When any of these termsare used with the “substituted” modifier one or more hydrogen atom hasbeen independently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —S(O)₂OH, or—S(O)₂NH₂. The groups —CH═CHF, —CH═CHCl and —CH═CHBr are non-limitingexamples of substituted alkenyl groups.

The term “alkynyl” when used without the “substituted” modifier refersto a monovalent unsaturated aliphatic group with a carbon atom as thepoint of attachment, a linear or branched acyclic structure, at leastone carbon-carbon triple bond, and no atoms other than carbon andhydrogen. As used herein, the term alkynyl does not preclude thepresence of one or more non-aromatic carbon-carbon double bonds. Thegroups —C≡CH, —C≡CCH₃, and —CH₂C≡CCH₃ are non-limiting examples ofalkynyl groups. An “alkyne” refers to the class of compounds having theformula H—R, wherein R is alkynyl. When any of these terms are used withthe “substituted” modifier one or more hydrogen atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂.

A “repeat unit” is the simplest structural entity of certain materials,for example, frameworks and/or polymers, whether organic, inorganic ormetal-organic. In the case of a polymer chain, repeat units are linkedtogether successively along the chain, like the beads of a necklace. Forexample, in polyethylene, —[—CH₂CH₂—]_(n)—, the repeat unit is —CH₂CH₂—.The subscript “n” denotes the degree of polymerization, that is, thenumber of repeat units linked together. When the value for “n” is leftundefined or where “n” is absent, it simply designates repetition of theformula within the brackets as well as the polymeric nature of thematerial. The concept of a repeat unit applies equally to where theconnectivity between the repeat units extends three dimensionally, suchas in metal organic frameworks, modified polymers, thermosettingpolymers, etc.

A “sugar moiety” is a monovalent naturally or unnatural saccharide whichis linked to the formula through a covalent bond between the formula anda heteroatom on the saccharide. Some non-limiting examples ofcarbohydrates which are included in the term sugar moiety include:glucose, galactose, xylose, ribose, arabinose, glyceraldehyde,erythrose, or mannose. The term may also include derivatized saccharidessuch as amino sugars or sulfosugars such as galactosamine, sialic acid,glucosamine, N-acetylglucosamine, or sulfoquinovose.

II. NANOPARTICLE COMPOSITIONS AND FORMULATIONS

A. Hydrophobic Compounds

In some aspects of the present disclosure, the nanoparticle compositioncomprises a mixture of hydrophobic compounds such as phospholipids,steroids such as cholesterols, and other triglycerides. In someembodiments, these hydrophobic compounds are formulated to mimic thecomposition of a high density lipoprotein (HDL). In some embodiments,the nanoparticle comprises 1, 2, 3, 4, or more different types ofhydrophobic compounds. Additionally, it is contemplated that thenanoparticle composition may comprise multiple different hydrophobiccompounds within one type (e.g. multiple different phospholipids ordifferent steroid derivatives). In some embodiments, the hydrophobiccompound is a steroid or a steroid derivative. In other embodiments, thehydrophobic compound is a phospholipid or mixture of phospholipids. Insome embodiments, the hydrophobic compound is a composition of two,three, or more phospholipids and one or more triglycerides. In otherembodiments, the nanoparticle compositions comprise a steroid or asteroid derivative and a mixture of different types of phospholipids.

In some aspects, the nanoparticle composition comprises from about 0.5w/w % to about 12.5 w/w % of a steroid or steroid derivative. The amountof steroid or steroid derivative may be from about 0.5 w/w %, 1 w/w %, 2w/w %, 3 w/w %, 3.5 w/w %, 4 w/w %, 4.5 w/w %, 5 w/w %, 5.5 w/w %, 6 w/w%, 6.5 w/w %, 7 w/w %, 8 w/w %, 9 w/w %, 10 w/w %, 11 w/w %, 12 w/w %,to about 12.5 w/w %, or any range derivable therein. In someembodiments, the steroid or steroid derivative comprises about 4.8 w/w %of the nanoparticle composition.

In some aspects, the nanoparticle composition comprises from about 10w/w % to about 45 w/w % of the phospholipid composition. The amount ofphospholipid composition may be from about 10 w/w %, 12.5 w/w %, 15 w/w%, 17.5 w/w %, 20 w/w %, 21 w/w %, 22 w/w %, 22.5 w/w %, 23 w/w %, 24w/w %, 25 w/w %, 27.5 w/w %, 30 w/w %, 32.5 w/w %, 35 w/w %, 37.5 w/w %,40 w/w %, 42.5 w/w %, to about 45 w/w %, or any range derivable therein.In some embodiments, the phospholipid composition comprises about 4.8w/w % of the nanoparticle composition.

1. Steroids and Steroid Derivatives

In some aspects of the present disclosure, the polymers are mixed withone or more steroid or a steroid derivative to create a nanoparticlecomposition. In some embodiments, the steroid or steroid derivativecomprises any steroid or steroid derivative. As used herein, in someembodiments, the term “steroid” is a class of compounds with a four ring17 carbon cyclic structure which can further comprises one or moresubstitutions including alkyl groups, alkoxy groups, hydroxy groups, oxogroups, acyl groups, or a double bond between two or more carbon atoms.In one aspect, the ring structure of a steroid comprises three fusedcyclohexyl rings and a fused cyclopentyl ring as shown in the formulabelow:

In some embodiments, a steroid derivative comprises the ring structureabove with one or more non-alkyl substitutions. In some embodiments, thesteroid or steroid derivative is a sterol wherein the formula is furtherdefined as:

In some embodiments of the present disclosure, the steroid or steroidderivative is a cholestane or cholestane derivative. In a cholestane,the ring structure is further defined by the formula:

As described above, a cholestane derivative includes one or morenon-alkyl substitution of the above ring system. In some embodiments,the cholestane or cholestane derivative is a cholestene or cholestenederivative or a sterol or a sterol derivative. In other embodiments, thecholestane or cholestane derivative is both a cholestere and a sterol ora derivative thereof.

2. Phospholipids

In some aspects of the present disclosure, the polymers are mixed withone or more phospholipids to create a nanoparticle composition. In someembodiments, any lipid which also comprises a phosphate group. In someembodiments, the phospholipid is a structure which contains one or twolong chain C6-C24 alkyl or alkenyl groups, a glycerol or a sphingosine,one or two phosphate groups, and, optionally, a small organic molecule.In some embodiments, the small organic molecule is an amino acid, asugar, or an amino substituted alkoxy group, such as choline orethanolamine. In some embodiments, the phospholipid is further definedby a compound of the formula:

wherein:

-   -   R₁ and R₂ are each independently alkyl_((C6-24)),        alkenyl_((C6-24)), or a substituted version of either of these        groups; and    -   R₃ is hydrogen or —(CH₂)_(x)R_(a), wherein:        -   x is 1, 2, 3, 4, 5, or 6; and        -   R_(a) is —NR′R″R′″⁺ or —CH(CO₂R_(b))NR_(c)R_(d), wherein:            -   R′, R″, and R′″ are each independently hydrogen,                alkyl_((C≦6)), or substituted alkyl_((C≦6)); and            -   R_(b), R_(c), and R_(d) are each independently hydrogen,                alkyl_((C≦6)), or substituted alkyl_((C≦6)); or                a compound of the formula:

wherein:

-   -   R₄ and R₅ are each independently alkyl_((C6-24)),        alkenyl_((C6-24)), or a substituted version of either of these        groups; and    -   R₆ is hydrogen or —(CH₂)_(x)R_(a), wherein:        -   x is 1, 2, 3, 4, 5, or 6; and        -   R_(a) is —NR′R″R′″⁺ or —CH(CO₂R_(b))NR_(c)R_(d), wherein:            -   R′, R″, and R′″ are each independently hydrogen,                alkyl_((C≦6)), or substituted alkyl_((C≦6)); and            -   R_(b), R_(c), and R_(d) are each independently hydrogen,                alkyl_((C≦6)), or substituted alkyl_((C≦6));    -   R₇ is hydroxy or alkoxy_((C≦6)), acyloxy_((C≦6)), or a        substituted version of either of these groups;        or salts thereof.

In some embodiments, the phospholipid is a phosphatidylcholine. In otherembodiments, the phospholipid is a phosphatidylserine. In otherembodiments, the phospholipid is a sphingomyelin. In some embodiments,the nanoparticle composition comprises a mixture of phospholipids toobtain a phospholipid composition such as a mixture ofphosphatidylserine, phosphatidylcholine, and sphingomyelin. In someembodiments, the phospholipid composition comprises a ratio of the firstphospholipid to the second phospholipid from about 10:1 to about 1:2. Insome embodiments; the ratio of the first phospholipid to the secondphospholipid is from about 10:1, 9:1, 8:1, 7.5:1, 7:1, 6.5:1, 6:1,5.5:1, 5:1, 4.5:1, 4:1, 3:1, 2:1, 1:1, to about 1:2, or any rangederivable therein. In some embodiments; the ratio is about 5.2:1. Insome embodiments, the nanoparticle composition comprises a thirdphospholipid. In some embodiments, the third phospholipid is present ina ratio to the first phospholipid from about 25:1 to about 1:1. In someembodiments, the ratio is from about 25:1, 24:1, 22:1, 20:1, 19:1, 18:1,17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 8:1, 6:1, 4:1, 2:1, toabout 1:1, or any range derivable therein. In some embodiments, theratio is about 15.7:1.

3. Triglycerides

In some aspects of the present disclosure, the nanoparticle compositionsmay further comprise one or more triglycerides. In various embodiments,the triglyceride is compound of the structure:

wherein:

-   -   R₁ and R₂ are each independently hydrogen or —C(O)—R₄; and    -   Y is hydrogen, hydroxy, a sugar moiety, or —OC(O)—R₄; wherein:        -   R₄ is an alkyl_((C1-25)), alkenyl_((C1-25)),            alkynyl_((C1-25)), or a substituted version of any of these            groups; or a group of the formula: —C(O)—X—C(O)H, wherein X            is an alkanediyl_((C1-12)) or substituted            alkanediyl_((C1-12));    -   provided that R₁, R₂, and Y are not all hydrogen.

In some embodiments, R₄ is selected from the group C₁-C₂₅ substituted orunsubstituted alkyl, C₁-C₂₅ substituted or unsubstituted alkenyl, C₁-C₂₅substituted or unsubstituted alkynyl, and —C(O)—X—C(O)H, wherein X is—(CH₂)_(z)—, wherein Z=1-12. In some embodiments, R₄ is selected fromthe group C₁-C₂₅ alkyl, C₁-C₂₅ alkenyl, C₁-C₂₅ alkynyl, and—C(O)—X—C(O)H, wherein X is —(CH₂)_(z)—, wherein Z=1-12. In the abovestructure it is important to note that if one or more of R₁ and R₂ are—C(O)—R₄ and/or Y is —OC(O)—R₄, then a different R₄ group may beassociated with R₁, R₂, and/or Y (e.g., R₁, R₂, and/or Y do not need tohave the same R₄ group). In other embodiments, the Y group is a sugarmoiety such as ether linked galactose, fructose, glucose, or xylose.

In some embodiments, R₁ or R₂ is —C(O)—R₄, wherein R₄ is C₄-C₁₈ alkyl,C₈-C₂₅ alkenyl, or C₈-C₂₅ alkynyl. In other embodiments, R₄ is—(CH₂)_(Y)—H, wherein Y=8-10. In some embodiments, R₁, R₂, and/or R₃ isa caprylic group, a capric group, a linoleic group, or a succinic group.In other embodiments, R₁ and R₂ are each independently an alkenyl groupof 8-24 carbon atoms. In some aspects, the triglyceride is a compositioncomprising two or more different triglyceride molecules.

4. Cholesterol

In some aspects of the present disclosure, the nanoparticle compositionsmay further comprise a specific steroid class of steroids calledcholesterol. Cholesterol has the formula:

It is contemplated that any stereoisomers of the cholesterol moleculeabove. Furthermore, the molecule could be saturated such that the doublebond in the B ring is hydrogenated to obtain a single bond. In otherembodiments, the hydroxyl group in the A ring can also be oxidized toobtain a carbonyl. If the A ring has been oxidized, the carbonyl canalso be an imino or thiocarbonyl group instead of an oxo group. In otherembodiments, the cholesterol molecule is the natural isomer with theformula:

B. Vitamin E Components

In some embodiments, the nanoparticle composition comprises α-tocopherolor a derivative of α-tocopherol such as α-tocopheryl acetate orsuccinate. These compounds may also be conjugated with additional groupsto add further functionalities. These additional groups include groupssuch as a hydrophobic group such as a fatty acid or long chain alkylgroup on the free carboxyl group. In other embodiments, the nanoparticlecomposition is a PEGylated tocopheryl succinate compound. In someembodiments, the PEGylated tocopheryl succinate comprises a tocopherolsuccinate of a formula:

and a PEGylated group attached to the free carboxyl group. In someembodiments, the PEG group comprises a repeating unit of ethylene glycolwith a number of repeating units from 1 to 1,000. PEG is the polymericform of ethylene glycol. The PEG portion of the compound has theformula:

Tocopheryl-(OCH₂CH₂)_(n)OH   (IV)

wherein the repeating unit, n, is an integer. The number of repeatingunits may be from about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300,400, 500, 600, 700, 800, 900, to about 1,000 units, or any rangederivable therein. In some aspects, the nomenclature used to describePEG includes the average molecular weight of the polymer (e.g. PEG-800;PEG-1000, PEG-1200, etc.). As would be obvious to a person of skill inthe art, the average molecular weight does not mean that any particularPEG component within the composition has the noted molecular weight butrather that the component as a whole has the average molecular weightcorresponding to that value. In some embodiments, the PEG component canhave a terminal hydrogen atom can be replaced with another groupincluding but not limited to a C₁-C₆ alkyl group (e.g. a methyl group oran ethyl group), or a reactive moiety used to attach the PEG to anothercompound. For example, a PEG-1000 composition generally comprises PEGmolecules with 16 and 17 repeating units as shown in the formula above,but may also comprises individual PEG molecules with less than 16 ormore than 17 repeating units. As the value in the name of the PEGcomponent represents the average molecular weight, the overall polymeraverage molecular weight may be modified to obtain an average molecularweight from less than 500 to over a 2500 g/mol (e.g. about 10 repeatingunits to about 40 repeating units). In some embodiments, the PEGcomponent of the molecule has an average molecular weight equal to orless than PEG-1000.

In some aspects, the present disclosure provides nanoparticles whichcomprises from about 5 w/w % to about 60 w/w % of the α-tocopherylcompound. In some embodiments, the α-tocopheryl compound comprise fromabout 5 w/w %, 10 w/w %, 11 w/w %, 12 w/w %, 13 w/w %, 14 w/w %, 15 w/w%, 16 w/w %, 17 w/w %, 18 w/w %, 19 w/w %, 20 w/w %, 25 w/w %, 30 w/w %,35 w/w %, 40 w/w %, 45 w/w %, 50 w/w %, 55 w/w %, to about 60 w/w %, orany range derivable therein.

C. Apolipoproteins

In some aspects, the nanoparticle compositions of the present disclosuremay comprise one or more apolipoprotein. Apolipoproteins are associatedwith lipid metabolism and are present in a variety of differentlipoproteins. These proteins are the major protein components oflipoproteins with Apolipoprotein A1 being the primary protein in highdensity lipoproteins. These proteins are associated with the transportof fat through the body. Without wishing to be bound by any theory, itis believed that these molecules bind to the outside of the lipidposition of the lipoproteins to increase the lipoproteins'watersolubility. Other apolipoprotein including apolipoprotein A-II,apolipoprotein A-IV, apolipoprotein A-V, apolipoprotein C such asapolipoproteins C-I, C-II, C-III, and C-IV, apolipoprotein D,apolipoprotein E, apolipoprotein H, and apolipoprotein L. In otherembodiments, the apolipoprotein is apolipoprotein B such asapolipoprotein B48 or apolipoprotein B100. In some aspects,apolipoproteins A, C, and E share similar genetic origins and may beused in similar applications. It is also contemplated that one of theseapolipoproteins may be modified such as through mutation or theattachment of a second compound or biologic component.

D. Therapeutic Agents

In some aspects, the nanoparticle compositions of the present disclosurecomprise one or more therapeutic agents. In some embodiments, thenanoparticles comprise 1, 2, 3, 4, or 5 therapeutic agents. In someembodiments, the nanoparticles comprise 1 therapeutic agent or 2therapeutic agents. In some aspects, the nanoparticle compositionscomprise from about 0.5 w/w % to about 25 w/w %. In some embodiments,the nanoparticle compositions comprise from about 0.5 w/w %, 1 w/w %, 2w/w %, 3 w/w %, 4 w/w %, 5 w/w %, 6 w/w %, 7 w/w %, 8 w/w %, 9 w/w %, 10w/w %, 11 w/w %, 12 w/w %, 13 w/w %, 14 w/w %, 15 w/w %, 17.5 w/w %, 20w/w %, 22.5 w/w %, to about 2.5 w/w %, or any range derivable therein.In some embodiments, the amount of therapeutic agent is about 3.8 w/w %of the composition. In other embodiments, the amount of the therapeuticagent is about 10 w/w % of the composition.

1. Nucleicids

In some aspects of the present disclosure, the nanoparticle compositionscomprise one or more nucleic acids. In addition, it should be clear thatthe present disclosure is not limited to the specific nucleic acidsdisclosed herein. Formulations of pro-ISNP compositions may furthercomprise a nucleic acid based therapeutic agents. The present disclosureis not limited in scope to any particular source, sequence, or type ofnucleic acid, however, as one of ordinary, skill in the art couldreadily identify related homologs in various other sources of thenucleic acid including nucleic acids from non-human species (e.g.,mouse, rat, rabbit, dog, monkey, gibbon, chimp, ape, baboon, cow, pig,horse, sheep, cat and other species). it is contemplated that thenucleic acid used in the present disclosure can comprises a sequencebased upon a naturally-occurring sequence. Allowing for the degeneracyof the genetic code, sequences that have at least about 50%, usually atleast about 60%, more usually about 70%, most usually about 80%,preferably at least about 90% and most preferably about 95% ofnucleotides that are identical to the nucleotide sequence of thenaturally-occurring sequence. In another embodiment, the nucleic acid isa complementary sequence to a naturally occurring sequence, orcomplementary to 75%, 80%, 85%, 90%, 95% and 100%.

In some aspects, the nucleic acid is a sequence which silences, iscomplimentary to, or replaces another sequence present in vivo.Sequences of 17 bases in length should occur only once in the humangenome and, therefore, suffice to specify a unique target sequence.Although shorter oligomers are easier to make and increase in vivoaccessibility, numerous other factors are involved in determining thespecificity of hybridization. Both binding affinity and sequencespecificity of an oligonucleotide to its complementary target increaseswith increasing length. It is contemplated that exemplaryoligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or morebase pairs will be used, although others are contemplated. Longerpolynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000 orlonger are contemplated as well.

As stated above, “complementary” or “antisense” means polynucleotidesequences that are substantially complementary over their entire lengthand have very few base mismatches. For example, sequences of fifteenbases in length may be termed complementary when they have complementarynucleotides at thirteen or fourteen positions. Naturally, sequenceswhich are completely complementary will be sequences which are entirelycomplementary, throughout their entire length and have no basemismatches. Other sequences with lower degrees of homology also arecontemplated. For example, an anti sense construct which has limitedregions of high homology, but also contains a non-homologous region(e.g., ribozyme; see below) could be designed. These molecules, thoughhaving less than 50% homology, would bind to target sequences underappropriate conditions.

Inhibitory RNA. As mentioned above, the present disclosure contemplatesthe use of one or more inhibitory nucleic acid for reducing expressionand/or activation of a gene or gene product. Examples of an inhibitorynucleic acid include but are not limited to molecules targeted to annucleic acid sequence, such as a microRNA, an siRNA (small interferingRNA), short hairpin RNA (shRNA), double-stranded RNA, an anti senseoligonucleotide, a ribozyme and molecules targeted to a gene or geneproduct such as an aptamer.

An inhibitory nucleic acid may inhibit the transcription of a gene orprevent the translation of the gene transcript in a cell. An inhibitorynucleic acid may be from 16 to 1000 nucleotides long, and in certainembodiments from 18 to 100 nucleotides long.

In some embodiment, an inhibitory nucleic acid is capable of decreasingthe expression of a particular genetic product by at least 10%, at least20%, at least 30%, or at least 40%, at least 50%, at least 60%, or atleast 70%, at least 75%, at least 80%, at least 90%, at least 95% ormore or any ranges in between the foregoing.

In some embodiments, the nucleic acids of the present disclosurecomprise one or more modified nucleosides comprising a modified sugarmoiety. Such compounds comprising one or more sugar-modified nucleosidesmay have desirable properties, such as enhanced nuclease stability orincreased binding affinity with a target nucleic acid relative to anoligonucleotide comprising only nucleosides comprising naturallyoccurring sugar moieties. In some embodiments, modified sugar moietiesare substituted sugar moieties. In some embodiments, modified sugarmoieties are sugar surrogates. Such sugar surrogates may comprise one ormore substitutions corresponding to those of substituted sugar moieties.In some embodiments, nucleosides of the present disclosure comprise oneor more unmodified nucleobases. In certain embodiments, nucleosides ofthe present disclosure comprise one or more modified nucleobases.

2. Peptides and Proteins

The use of peptides and proteins as drugs continues to grow. As withmany complex molecules, delivery issues may prevent the effective use ofpeptide/polypeptide drugs. Thus, the nanoparticles of the presentdisclosure may find use for the delivery of peptide/polypeptide drugsincluding but not limited to antibodies (Infliximab, Herceptin,Cetuximab, Rituximab), peptide hormones (insulin), clotting factors,anti-cancer peptides (Adalimumab, Aflibercept, Alemtuzumab, Bevacizumab,Bortezomib, Cilengitide, Triptorelin pamoate, Leuprolide acetate,Histrelin acetate, Goserelin acetate, Buserelin acetate, Abarelixacetate, Degarelix acetate), cytokines, interferons, interleukins IL-2,etc.), antivirals (Enfuvirtide), growth factors, enzymes (TPA), and ahost of others (Teriparatide, Exenatide, Liraglutide, Lanreotide,Pramlintide, Ziconotide, Icatabant, Ecallantide, Tesamorelin,Mifamurtide and Nesiritude).

i. Therapeutic Antibodies

In some aspects, the nanoparticle compositions may further comprise anantibody or a fragment thereof that binds to at least a portion of anantigen are contemplated. As used herein, the term “antibody” isintended to refer broadly to any immunologic binding agent, such as IgG,IgM, IgA, IgD, IgE, and genetically modified IgG as well as polypeptidescomprising antibody CDR domains that retain antigen binding activity.The antibody may be selected from the group consisting of a chimericantibody, an affinity matured antibody, a polyclonal antibody, amonoclonal antibody, a humanized antibody, a human antibody, or anantigen-binding antibody fragment or a natural or synthetic ligand.

Thus, by known means and as described herein, polyclonal or monoclonalantibodies, antibody fragments, and binding domains and CDRs (includingengineered forms of any of the foregoing) may be created that arespecific to the antigen, one or more of its respective epitopes, orconjugates of any of the foregoing, whether such antigens or epitopesare isolated from natural sources or are synthetic derivatives orvariants of the natural compounds. Another variation is the constructionof bispecific antibodies in which one heavy chain targeting one antigenand other heavy chain targeting a different antigen.

Examples of antibody fragments suitable for the present embodimentsinclude, without limitation: (i) the Fab fragment, consisting of V_(L),V_(H), C_(L), and C_(H1) domains; (ii) the “Fd” fragment consisting ofthe V_(H) and C_(H1) domains; (iii) the “Fv” fragment consisting of theV_(L) and V_(H) domains of a single antibody; (iv) the “dAb” fragment,which consists of a V_(H) domain; (v) isolated CDR regions; (vi) F(ab′)2fragments, a bivalent fragment comprising two linked Fab fragments;(vii) single chain Fv molecules (“scFv”), wherein a V_(H) domain and aV_(L) domain are linked by a peptide linker that allows the two domainsto associate to form a binding domain; (viii) bi-specific single chainFv dimers (see U.S. Pat. No. 5,091,513); and (ix) diabodies, multivalentor multispecific fragments constructed by gene fusion (US Patent App.Pub. 20050214860). Fv, scFv, or diabody molecules may be stabilized bythe incorporation of disulphide bridges linking the V_(H) and V_(L)domains. Minibodies comprising a scFv joined to a CH₃ domain may also bemade (Hu, et al., 1996).

Antibody-like binding peptidomimetics are also contemplated inembodiments. Liu et al. (2003) describe “antibody like bindingpeptidomimetics” (ABiPs), which are peptides that act as pared-downantibodies and have certain advantages of longer serum half-life as wellas less cumbersome synthesis methods. (Liu; et al., 2003).

ii. Protein Therapeutics

In some embodiments, the nanoparticle compositions may comprise orcontain a therapeutic protein. The therapeutic protein may be a naturaland nonnatural (e.g., recombinant) proteins, polypeptides, and peptides.The proteins may, by themselves, be incapable of passing (or which passonly a fraction of the administered dose) through the gastrointestinalmucosa or may be susceptible to chemical cleavage by acids or enzymes inthe gastrointestinal tract or both. In addition to proteins, thenanoparticle composition also may include polysaccharides, andparticularly mixtures of mucopolysaccharides, carbohydrates, lipids;other organic compounds.

Examples of proteins that may be comprised in a hydrogel copolymer ofthe present invention include, but are not limited to, synthetic,natural, or recombinant sources of: a growth hormone-releasing hormone,an interleukin (e.g., IL-1 beta); a growth factor (e.g., STEMGEN®(ancestim; stem cell factor); a basic fibroblast growth factor (e.g.,high molecular weight FGF-2), a hepatocyte growth factor; erythropoietin(e.g., PROCRIT®, EPREX®, or EPOGEN® (epoetin-α); ARANESP®(darbepoetin-α); NEORECORMON®, EPOGIN® (epoetin-β); and the like); ablood factor (e.g., ACTIVASE® (alteplase) tissue plasminogen activator;NOVOSEVEN® (recombinant human factor VIIa); Factor VIIa; Factor VIII(e.g., KOGENATE®); Factor IX (e.g., BENEFIX®, RIXUBIS™, ALPROLIX™);hemoglobin; and the like); an antigen; a soluble receptor (e.g., aTNF-α-binding soluble receptor such as ENBREL® (etanercept); a solubleVEGF receptor; a soluble interleukin receptor; a soluble γ/δ T cellreceptor; and the like); an enzyme (e.g., α-glucosidase; CERAZYME®(imiglucarase; β-glucocerebrosidase, CEREDASE® (alglucerase); an enzymeactivator (e.g., tissue plasminogen activator); an angiogenic agent(e.g., vascular endothelial growth factor (VEGF); an anti-angiogenicagent (e.g., a soluble VEGF receptor); thrombopoietin; glial fibrillaryacidic protein; a follicle stimulating hormone; a human alpha-1antitrypsin; a leukemia inhibitory factor; a transforming growth factor;a tissue factor; a macrophage activating factor, a neutrophilchemotactic factor; fibrin; a leukemia inhibitory factor; or a proteaseinhibitor (e.g., β₂-macroglobulin). Combinations, analogs, fragments,mimetics or polyethylene glycol (PEG)-modified derivatives of thesecompounds, or other derivatives of any of the above-mentioned substancesmay also be suitable. Also suitable for use are fusion proteinscomprising all or a portion of any of the foregoing proteins. One ofordinary skill in the art, with the benefit of the present disclosure,may recognize additional drugs, including drugs other than proteins,which may be useful in the compositions and methods of the presentdisclosure. Such drugs are still considered to be within the spirit ofthe present disclosure.

a. Growth Factors

In some embodiments, the present disclosure includes nanoparticlecompositions which contain nerve growth factor (NGF) which may includeany form of biologically active nerve growth factor including the βsubunit of human nerve growth factor. The nerve growth factor may alsoinclude hybridized and modified forms of NGF which bind to the NGFreceptor and retain NGF bioactivity. Modified forms of NGF may alsoinclude fusion proteins such as, for example, Iwai, et al., 1986 andKanaya, et al., 1989, and NGF fragments and hybrids in which certainamino acids have been deleted or replaced while maintaining NGFbioactivity and receptor binding.

In some embodiments, the nanoparticle compositions with NGF containhuman NGF (hNGF) including recombinant hNGF (rhNGF). Methods ofpreparing NGF are known in the art and include, for example, abaculovirus expression system (Barnett, et al., 1990), a yeastexpression system (Kanaya, et al., 1989), a mammalian cell (CHO)expression system (Iwane, et al. 1990), a COS expression system (Bruce,et al., 1989), or bacterial expression system (Iwai, et al., 1986). TheNGF which may be used herein includes NGF which is greater than 65%pure. In some embodiments, the NGF is greater than 85% pure. In someembodiments, the NGF is greater than 95% pure. In some embodiments, theNGF is greater than 98% pure. The purity may be determined bysilver-stained SDS-PAGE or other means known to those skilled in theart.

In addition to NGF, other therapeutic agents include but not limited topigmented epithelial derived factor (PEDF), basic fibroblast growthfactor (bFGF), ciliary neurotrophic factor (CNTF). These therapeuticagents may be encapsulated into the nanoparticles, including thoseformulated for administration to the eyes. NGF, PEDF, bFGF, and CNTFhave been demonstrated to be protective against various retinopathies inin vitro and in vivo models of ocular diseases, such as, glaucoma,age-related macular degeneration, diabetic retinopathy, retinal ischemicabnormalities, uveitis, optic nerve trauma, endophthalmitis and otherocular diseases.

3. Small Molecules

The overwhelming majority of drugs—antibiotics, antiviral, cancerchemotherapeutics, anti-hypertensives, statins, anti-depressives, andmany others—and many others are categorized as “small molecules,” ageneral term applied to the class of compounds also described asorganopharmaeuticals. In some aspects, these drugs or therapeutic agentsare compounds which have a molecular weight of less than 2500 g/mol. Insome embodiments, the therapeutic agents have a molecular weight fromabout 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 900, 1000, 1250, 1500, 1750, 2000, 2250, to about 2500 g/mol.These therapeutic agents may be compounds which have a definitivestructural formula and may be present as a neutral molecule or as asalt. In some embodiments; small molecule therapeutic agents arecompounds which have a definitive chemical structure and formula whichexpressed through a specific connectivity of bonds and atoms. In anotherembodiment, the therapeutic agents used in the methods described hereinare small molecule compounds which are not particular soluble in water.Some non-limiting examples of therapeutic agents are BCS classes II andIV compounds or other agents that similarly exhibit poor solubility. TheBCS definition describes a compound in which the effective dosing is notsoluble in 250 mL of water at a pH from 1-7.5. The USP categories “veryslightly soluble” and “insoluble” describe a material that requires1,000 or more parts of the aqueous liquid to dissolve 1 part solute. Asused herein, when a compound is described as poorly soluble, it refersto a compound which has solubility in water of less than 1 mg/mL.

The methods of the present disclosure may be used to preparenanoparticles using many classes of therapeutic agents including, butnot limited to chemotherapeutics, agents for the prevention ofrestenosis, agents for treating renal disease; agents used forintermittent claudication, agents used in the treatment of hypotensionand shock, angiotensin converting enzyme inhibitors; antianginal agents,anti-arrhythmics, anti-hypertensive agents, antiotensin ii receptorantagonists, antiplatelet drugs, β-blockers β1 selective, beta blockingagents, botanical products for cardiovascular indications, calciumchannel blockers, cardiovascular/diagnostics, central alpha-2 agonists,coronary vasodilators, diuretics and renal tubule inhibitors, neutralendopeptidase/angiotensin converting enzyme inhibitors, peripheralvasodilators; potassium channel openers, anticonvulsants, antiemetics,antinauseants, anti-parkinson agents, antispasticity agents, cerebralstimulants, drugs to treat head trauma, drugs to assist with memory(e.g., to treat alzheimers/senility/dementia), drugs to treat migraine,drugs to treat movement disorders; also included are drugs to treat adisease such as multiple sclerosis, narcolepsy/sleep apnea, stroke,tardive dyskinesia; chronic graft versus host disease, eating disorders,learning disabilities, minimal brain dysfunction, obsessive compulsivedisorder, panic, alcoholism, drug abuse, developmental disorders,diabetes; benign prostate disease, sexual dysfunction, rejection oftransplanted organs, xerostomia, aids patients with kaposi's syndrome;antineoplastic hormones, biological response modifiers for cancertreatment; also included are vascular agents, cytoxic alkylating agents;cytoxic antimetabolics, cytoxics, immunomodulators, multi-drugresistance modulators, radiosensitizers, anorexigenic agents/CNSstimulants, antianxiety agents/anxiolytics, antidepressants,antipsychotics/schizophrenia, antimanics, sedatives and hypnotics,enkephalin analgesics, hallucinogenic agents, narcoticantagonists/agonists/analgesics, analgesics, epidural and intrathecalanesthetic agents, general, local, regional neuromuscular blockingagents sedatives, preanesthetic adrenal/acth, anabolic steroids,dopamine agonists, growth hormone and analogs, hyperglycemic agents,hypoglycemic agents, large volume parenterals (lvps), lipid-alteringagents, nutrients/amino acids, nutritional lvps, obesity drugs(anorectics), somatostatin, thyroid agents, vasopressin, vitamins otherthan d, anti allergy nasal sprays, antiasthmatic dry powder inhalers,antiasthmatic metered dose inhalers, antiasthmatics (nonsteroidal),(antihistamines, antitussives, decongestants, etc.), beta-2 agonists,bronchoconstrictors, bronchodilators, cough-cold-allergy preparations,inhaled corticosteroids, mucolytic agents, pulmonary anti-inflammatoryagents, pulmonary surfactants, anticholinergics, antidiarrheals,antiemetics, cathartics and laxatives, cholelitholytic agents,gastrointestinal motility modifying agents, h₂ receptor antagonists,inflammatory bowel disease agents, irritable bowel syndrome agents,liver agents, metal chelators, miscellaneous gastric secretory agents,miscellaneous gi drugs (including hemorrhoidal preparations),pancreatitis agents, pancreatic enzymes, prostaglandins, prostaglandins,gi, proton pump inhibitors, sclerosing agents, sucralfate,anti-progestins, contraceptives, oral contraceptives, estrogens,gonadotropins, gnrh agonists, gnrh antagonists, oxytocics, progestins,uterine-acting agents, anti-anemia drugs, anticoagulants,antifibrinolytics, antiplatelet agents, antithrombin drugs, coagulants,fibrinolytics, hematology, heparin inhibitors (including protaminesulfate & heparinase), blood drugs (e.g., drugs for hemoglobinopathies,hrombocytopenia, and peripheral vascular disease), prostaglandins,vitamin k, anti-androgens, androgens/testosterone, gnrh agonists, gnrhantagonists, aminoglycosides, antibacterial agents, sulfonamides,antibiotics, anti gonorrheal agents, anti-resistant antimicrobials,antisepsis immunomodulators, antitumor agents, cephalosporins,clindamycins, dermatologics, detergents, erythromycins, macrolides,anti-infectives (topical), other systemic antimicrobial drugs,otic-antibiotic in combination, penem antibiotics, penicillins, peptidesantibiotic, sulfonamides, systemic antibiotics, immunomodulators,immunostimulatory agents, aminoglycosides, anthelmintic agents,antibacterial (bacterial vaginosis), antibacterial quinolones,antifungal (candidiasis), antifungal, systemic,anti-infectives/systemic, antimalarials, antimycobacterial,antiparasitic agents, antiprotozoal agents, antitrichomonads,antituberculosis, chronic fatigue syndrome, immunomodulators,immunostimulatory agents, macrolides, other drugs-aids relatedillnesses, other antiparasitic antimicrobial drugs, spiramycin, systemicantibiotics anti-gout drugs, corticosteroids, systemic, cyclooxygenaseinhibitors, enzyme blockers, immunomodulators for rheumatic diseases,metalloproteinase inhibitors, nonsteroidal anti-inflammatory agents,non-steroidal anti-inflammatory agents, antifungals, antihistamines,contraceptives, detergents, non-narcotic analgesics, nsaids, vitamins,analgesics, nonnarcotic, antipyretics, counterirritants, musclerelaxant, anticaries preparations, antigingivitis agents, antiplaqueagents, antifibrinolytics, chelating agents, alpha adrenergicagonists/blockers, antibiotics, antifungals, antiprotozoals, antivirals,beta adrenergic blockers, carbonic anhydrase inhibitors,corticosteroids, immune system regulators, mast cell inhibitors,nonsteroidal anti-inflammatory agents, prostaglandins, and proteolyticenzymes.

III. FORMULATIONS AND THERAPEUTIC APPLICATIONS

A. Therapeutic Formulations

In some embodiments, the nanoparticles may be formulated as apharmaceutical or therapeutic composition appropriate for the intendedapplication. In certain embodiments, pharmaceutical compositions maycomprise, for example, at least about 0.1% of the nanoparticlecomposition. In other embodiments, the nanoparticle composition maycomprise between about 2% to about 75% of the weight of the unit, orbetween about 25% to about 60%, for example, and any range derivabletherein.

The therapeutic compositions of the present embodiments are administeredin the form of injectable compositions either as liquid solutions orsuspensions; solid forms suitable for solution in, or suspension in,liquid prior to injection may also be prepared. These preparations alsomay be emulsified.

The phrases “pharmaceutical or pharmacologically acceptable” refers tomolecular entities and compositions that do not produce an adverse,allergic, or other untoward reaction when administered to an animal,such as a human, as appropriate. The preparation of a pharmaceuticalcomposition comprising an antibody or additional active ingredient willbe known to those of skill in the art in light of the presentdisclosure. Moreover; for animal (e.g., human) administration, it willbe understood that preparations should meet sterility, pyrogenicity,general safety, and purity standards as required by FDA Office ofBiological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall aqueous solvents (e.g., water, alcoholic/aqueous solutions, salinesolutions, parenteral vehicles, such as sodium chloride, Ringer'sdextrose, etc.), non-aqueous solvents (e.g., propylene glycol,polyethylene glycol, vegetable oil, and injectable organic esters, suchas ethyloleate), dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial or antifungal agents, anti-oxidants,chelating agents, and inert gases), isotonic agents, absorption delayingagents, salts, drugs, drug stabilizers, gels, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, fluid and nutrient replenishers, such like materials andcombinations thereof, as would be known to one of ordinary skill in theart. The pH and exact concentration of the various components in apharmaceutical composition are adjusted according to well-knownparameters.

The term “unit dose” or “dosage” refers to physically discrete unitssuitable for use in a subject, each unit containing a predeterminedquantity of the therapeutic composition calculated to produce thedesired responses discussed above in association with itsadministration, i.e., the appropriate route and treatment regimen. Thequantity to be administered, both according to number of treatments andunit dose, depends on the effect desired. The actual dosage amount of acomposition of the present embodiments administered to a patient orsubject can be determined by physical and physiological factors, such asbody weight, the age, health, and sex of the subject, the type ofdisease being treated, the extent of disease penetration, previous orconcurrent therapeutic interventions, idiopathy of the patient, theroute of administration, and the potency, stability, and toxicity of theparticular therapeutic substance. For example, a dose may also comprisefrom about 1 μg/kg/body weight to about 1000 mg/kg/body weight (suchrange includes intervening doses) or more per administration, and anyrange derivable therein. In non-limiting examples of a derivable rangefrom the numbers listed herein, a range of about 5 μg/kg/body weight toabout 100 mg/kg/body weight, about 5 μg/kg/body weight to about 500mg/kg/body weight, etc., can be administered. The practitionerresponsible for administration will, in any event, determine theconcentration of active ingredient(s) in a composition and appropriatedose(s) for the individual subject.

Single or multiple doses of the agents are contemplated. Desired timeintervals for delivery of multiple doses can be determined by one ofordinary skill in the art employing no more than routineexperimentation. As an example, subjects may be administered two dosesdaily at approximately 12 hour intervals. In some embodiments, the agentis administered once a day.

The agent(s) may be administered on a routine schedule. As used herein aroutine schedule refers to a predetermined designated period of time.The routine schedule may encompass periods of time which are identicalor which differ in length, as long as the schedule is predetermined. Forinstance; the routine schedule may involve administration twice a day,every day, every two days, every three days, every four days, every fivedays, every six days, a weekly basis, a monthly basis or any set numberof days or weeks there-between. Alternatively, the predetermined routineschedule may involve administration on a twice daily basis for the firstweek, followed by a daily basis for several months, etc. In otherembodiments, the invention provides that the agent(s) may be takenorally and that the timing of which is or is not dependent upon foodintake. Thus, for example, the agent may be taken every morning and/orevery evening, regardless of when the subject has eaten or will eat.

The active compounds can be formulated for parenteral administration;e.g., formulated for injection via the intravenous, intramuscular,sub-cutaneous, or even intraperitoneal routes. Typically, suchcompositions can be prepared as either liquid solutions or suspensions;solid forms suitable for use to prepare solutions or suspensions uponthe addition of a liquid prior to injection can also be prepared; and;the preparations can also be emulsified.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil, or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that it may be easily injected. It also should be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi.

The therapeutically compositions may be formulated into a neutral orsalt form. Pharmaceutically acceptable salts, include the acid additionsalts (formed with the free amino groups of the protein) and which areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic; oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike. Additionally, the pharmaceutical or therapeutic compositions maycomprises one or more polycationic peptides or proteins such asprotamine, polylysine, or polyarginine such that the therapeutic agentis formulated as a neutral salt or as a complex which containssignificantly reduced charge.

A pharmaceutical composition can include a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating, such as lecithin,by the maintenance of the required particle size in the case ofdispersion, and by the use of surfactants. The prevention of the actionof microorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

The therapeutic compound may also be administered topically to the skin,eye, ear, or mucosal membranes. Administration of the therapeuticcompound topically may include formulations of the compounds as atopical solution, lotion, cream, ointment, gel, foam, transdermal patch,or tincture. When the therapeutic compound is formulated for topicaladministration, the compound may be combined with one or more agentsthat increase the permeability of the compound through the tissue towhich it is administered. In other embodiments, it is contemplated thatthe topical administration is administered to the eye. Suchadministration may be applied to the surface of the cornea, conjunctiva,or sclera. Without wishing to be bound by any theory, it is believedthat administration to the surface of the eye allows the therapeuticcompound to reach the posterior portion of the eye. Ophthalmic topicaladministration can be formulated as a solution, suspension, ointment,gel, or emulsion.

IV. KITS

The present disclosure also provides kits. Any of the componentsdisclosed herein may be combined in the form of a kit. In someembodiments, the kits comprise a nanoparticle composition as describedabove or in the claims.

The kits will generally include at least one vial, test tube, flask,bottle, syringe or other container, into which a component may beplaced, and preferably, suitably aliquoted. Where there is more than onecomponent in the kit, the kit also will generally contain a second,third or other additional containers into which the additionalcomponents may be separately placed. However, various combinations ofcomponents may be comprised in a container. In some embodiments, all ofthe delivery components are combined in a single container. In otherembodiments, some or all of the delivery components with the instantnanoparticle compositions are provided in separate containers.

The kits of the present disclosure also will typically include packagingfor containing the various containers in close confinement forcommercial sale. Such packaging may include cardboard or injection orblow molded plastic packaging into which the desired containers areretained or a glass vial containing a syringable composition. A kit mayalso include instructions for employing the kit components. Instructionsmay include variations that can be implemented.

V. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many, changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Nerve Growth Factor Nanoparticles to Cross the Blood-BrainBarrier

The inventors aimed to develop novel HDL-mimicking α-tocopherol-coatednerve growth factor (NGF) nanoparticles targeting scavenger receptorclass B type I (SR-BI) to cross the blood-brain barrier (BBB). Taguchiarray was used to assist the NP development. Different ion-pair agentswere employed to form an optimal ion-pair with NGF in order tofacilitate the encapsulation of NGF. The novel HDL-mimickingα-tocopherol-coated NGF NPs were fully characterized in terms ofparticle size, entrapment efficiency and Apo A-I loading.

Materials and Cell Culture. Protamine from salmon, protamine grade X,protamine sodium salt USP, poly-lysine and cholesteryl Oleate (CO) werepurchased from Sigma-Aldrich (St. Louis, Mo.). Sephadex G-50, SephadexG-100, Sephacryl S-100 and Sepharose CL-4B were also purchased fromSigma-Aldrich (St. Louis, Mo.). PC, SM, and phosphatidylserine (PS) werepurchased from Avanti polar lipids (Alabaster, Ala.). TPGS was providedby BSAF as a gift. Apo A-I was purchased from Athens research andtechnology (Athens, Ga.). Recombinant human NGF was purchased fromCreative Biomart (Shirley, N.Y.). Neurite outgrowth staining kit waspurchased from Molecular Probes by Life Technologies (Madison, Wis.).Bradford reagent was obtained from thermo scientific (Rockford, Ill.).Amicon ultra centrifugal filters-0.5 ml was obtained from Merk Millipore(Germany).

Optimization of preparation procedure for prototype HDL-mimicking NPs.Blank HDL-mimicking NPs were prepared by a self-assembly method. Allexcipients were dissolved in ethanol to prepare stock solutions. Certainamounts of PC (43.1%), SM (8.1%), PS (2.7%), CO (7.7%) and TPGS (38.4%)(percentages based on w/w) were added into a glass vial to form a thinfilm after removing ethanol by nitrogen. And then 1 ml of milliq waterwas added into the vial. Five different procedures were evaluated tohydrate the film to form NPs, including: 1) adding water at 50° C. andstifling at 50° C. for 30 min at 600 rpm, 2) adding water at 50° C. andstirring at room temperature (RT) for 30 min at 600 rpm, 3) adding waterat RT and stirring at RT for 30 min at 600 rpm, and 4) adding water at50° C. and homogenizing 5 min using a homogenizer at 8600 rpm, and 5)adding water at RT and homogenizing 5 min using a homogenizer at 8600rpm. To further evaluate the influence of homogenization time on NPformation, the mixtures were homogenized for 0, 1, 2, 3, 4, 5, and 6 minafter adding water at RT. After preparation, particle size andpolydispersity index (P.I.) of NPs were measured using a Delsa Nano HCparticle analyzer (Beckman Coulter, Calif.) at 90° light scattering at25° C.

Development of Prototype HDL-Mimicking NPs by Taguchi Array

Taguchi array for NPs without Apo A-I. PC, SM and PS were selected asphospholipid components and CO was selected as the lipid component todevelop the HDL-mimicking NPs. To simplify the design and quickly findthe optimal compositions, the inventors considered phospholipids as onevariable. The percentage of each phospholipid was fixed as PC (78%), SM(14%) and PS (3%) in the total phospholipids, which is close to thecomposition of phospholipids in natural HDLs. To evaluate differentratios of phospholipids and CO, the inventors designed two Taguchiarrays. In Taguchi array #1 (Table 2A and 2B), the ratio ofphospholipids and CO was controlled around 1:1 (phospholipids/CO, w/w).Taguchi array for 3 levels 2 variables (phospholipids and CO) was usedto give three different concentrations for each excipient. In Taguchiarray #2 (Table 2C and 2D), an array for 2 levels 2 variables was usedto give the ratio of phospholipids and CO around 4:1 to 8:1(phospholipids/CO, w/w), NPs were prepared as described above. Afterforming the thin film, 1 ml of milliq water at RT was added into thevial and homogenized for 5 min to form NPs. To make TPGS-coated NPs,certain amounts of TPGS were added into Taguchi array (Table 2C and 2D)to give a total surfactant (phospholipids+TPGS) within 60 μg/ml to 110μg/ml. Particle size and P.I. were measured as described above.

-   -   Tables 2A-2D. Taguchi array for development of HDL-mimicking        α-tocopherol-coated NPs. Listed are the compositions per 1 ml        NPs. A: Taguchi array with high contents of CO without TPGS, B:        modified 2A by adding TPGS into the compositions, C: Taguchi        array with low contents of CO without TPGS, and D: modified 2C        by adding TPGS into the composition.

2A. Exper- PC SM PS CO Particle size iment (μg) (μg) (μg) (μg) (nm) P.I.1-1 32 6 4 40 275.4 0.265 1-2 32 6 4 50 383.4 0.181 1-3 32 6 4 60 242.90.295 1-4 40 7.5 5 40 284.1 0.31 1-5 40 7.5 5 50 333.6 0.301 1-6 40 7.55 60 404.1 0.193 1-7 48 9 6 40 386.4 0.284 1-8 48 9 6 50 282.6 0.297 1-948 9 6 60 255.2 0.255

2B. Exper- PC SM PS CO TPGS Particle size iment (μg) (μg) (μg) (μg) (μg)(nm) P.I. 2-1 32 6 4 40 60 173 0.261 2-2 32 6 4 50 40 181.9 0.236 2-3 326 4 60 20 198.7 0.223 2-4 40 7.5 5 40 30 173.1 0.263 2-5 40 7.5 5 50 40190 0.239 2-6 40 7.5 5 60 20 166 0.27 2-7 48 9 6 40 30 173.1 0.271 2-848 9 6 50 10 202 0.28 2-9 48 9 6 60 20 211.4 0.294

2C. Exper- PC SM PS CO Particle size iment (μg) (μg) (μg) (μg) (nm) P.I.3-1 40 7.5 2.5 5 246.6 0.312 3-2 40 7.5 2.5 10 301.7 0.307 3-3 56 10.53.5 5 269.2 0.234 3-4 56 10.5 3.5 10 296.1 0.332

2D. Exper- PC SM PS CO TPGS Particle size iment (μg) (μg) (μg) (μg) (μg)(nm) P.I. 4-1 40 7.5 2.5 5 30 192.7 0.259 4-2 40 7.5 2.5 10 30 178.90.283 4-3 56 10.5 3.5 5 50 171.6 0.295 4-4 56 10.5 3.5 10 50 162 0.268

Optimization of loading Apo A-I in the prototype HDL-mimicking NPs.Based on the particle size and size distribution, the optimalcompositions were selected to load Apo A-I, which are bolded in Tables2B and 2D. To load Apo A-I on NPs, after homogenization for 5 min asdescribed above, certain amounts of Apo A-I were added into eachcomposition (Table 3). Different conditions were evaluated to load ApoA-I, including 2-hour stirring at RT, 4-hour stirring at RT, 4-hourstirring at RT followed with incubation at 4° C. overnight, and 4-hourstirring at RT followed with stirring at 4° C. overnight. Particle sizeand size distribution were measured as described above. Entrapmentefficiency of Apo A-I was analyzed by ultrafiltration. Briefly, 0.2 mlof the NPs were added into Amicon Ultra (Molecular cutoff 100 KDa) andcentrifuged at 14000 rpm at 4° C. for 3 min. After this, 400 μl waterwere added into the insert of Amicon to wash the membrane with the samecentrifugation condition. Apo A-I was passed the membrane and washedwith the same approach as described above to measure the recovery of ApoA-I in this separation method. The concentration of unloaded (free) ApoA-I in the filtrate was measured by Bradford assay. Loading andentrapment efficiency of Apo A-I were calculated as follows:

% loading=(drug added into NP)/(total weight of excipients)×100%  Equation (1)

% entrapment efficiency=(1−unloaded drug/total drug added into NP)×100%  Equation (2)

Furthermore, detailed studies on Apo A-I loadings were performed basedon the composition of the batch 4-2. To optimize Apo A-I loading,different amounts of Apo A-I were added into the NPs (Table 4) bychanging the amount of PC, but keeping the same amounts of SM, PS, COand TPGS in the batch 4-2. Loading and entrapment efficiency of Apo A-Iwere measured and calculated as described above.

TABLE 3 Characterization of the prototype HDL-mimicking α-tocopherol-coated NPs. Each batch (experiment) contained the same composition asthe corresponding batch in Table 2, except for the addition of Apo A-I.Theoretical Exper- loading iment Apo Particle of Apo EE % Exper- numberin A-I size A-I of Apo iment Table 2 (μg) (nm) P.I. (mole %) A-I 5-1 1-670 194.2 0.273 1.5 8 5-2 2-4 80 256.7 0.264 1.9 12 5-3 2-6 70 177.80.291 1.4 16 5-4 2-7 70 152 0.253 1.5 18 5-5 3-3 80 251.7 0.33 2.8 5 5-64-2 70 148.5 0.26 2.43 26 5-7 4-3 80 173.8 0.305 2.12 20

TABLE 4 Influence of Apo A-I loading on the prototype HDL- mimickingα-tocopherol-coated NPs (n = 3). Batch 5-6 in Table 3 was modified bychanging the contents of PC and Apo A-I to obtain batch 5-8 and 5-9.Loading of Apo Apo Particle A-I EE % Exper- PC A-I size (%, of Apo iment(μg) (μg) (nm) P.I. w/w) A-I 5-6 40 70 145 ± 5 0.289 ± 0.012 43.8 31 ±5.4 5-8 39 106 152 ± 5 0.265 ± 0.012 54.3 31 ± 3.6 5-9 38 140  156 ± 110.273 ± 0.001 61.4 26 ± 2.5

Particle size stability of prototype HDL-mimicking NPs at 4° C. Thephysical stability of the prototype HDL-mimicking NPs was assessed overtime at 4° C. Prior to particle size measurement, nanoparticles wereallowed to equilibrate to RT. One ml of NPs was used to measure theparticle size and PI as described above.

Development of NGF-Loaded HDL-Mimicking NPs

Optimization of ion-pair complex for NGF. To efficiently load NGF intothe NPs, poly-lysine and three types of protamines were tested to forman ion-pair complex with NGF. Protamines included protamine from salmon,protamine grade X and protamine sodium salt USP. Poly-lysine, protaminesand NGF were dissolved in water at the concentration of 1 mg/ml. NGF wasadded into poly-lysine or protamine solutions at 0.8:1, 1:1, and 1:1.2ratios (NGF:polymer, w/w). The complex was allowed to stand at RT for 10min, and then diluted with 1 ml of water or PBS to measure particle sizeas described above and also to measure zeta potential using a Delsa NanoHC particle analyzer (Beckman Coulter, Calif.). The optimal ratio of thecomplex was determined according to particle size and zeta potential.

Preparation of NGF-loaded HDL-mimicking NPs. Poly-lysine and protamineUSP were selected to prepare NU-loaded NPs. Briefly, 10 μg of NGF wasmixed with 10 μg poly-lysine or protamine USP and kept for 10 min toform the complex. PC, SM, PS, CO and TPGS ethanol solutions (Table 6below) were mixed and then ethanol was removed by nitrogen to form thethin film as described above. Two procedures were tested to add the NGFcomplex into NPs. In the first procedure, the NGF complex was added intothe thin film, and then 1 ml of water at RT was added and homogenizedfor 5 min. In the second procedure, 1 ml of water at RT was first addedinto the thin film and homogenized for 5 min, and then the NGF complexwas added into the solution. After addition of the NGF complex, thesolution was incubated at 37° C. for 30 min, and then stirred at RT for30 min. After cooling, the defined amount of Apo A-I was added into thesolution and stirred at RT overnight to form the final NGF-loadedHDL-mimicking α-tocopherol-coated NPs. Particle size and zeta potentialwere measured as described above.

TABLE 6 The composition of the final HDL-mimicking α-tocopherol-coatedNGF NPs. Apo Cationic Unit PC SM PS CO TPGS A-I polymer NGF μg 59 11 415 45 159 10 10 w/w % 18.8 3.6 1.2 4.8 14.4 50.8 3.2 3.2

Determination of NGF entrapment Efficiency in NGF-loaded HDL-mimickingNPs. Gel filtration chromatography was used to separate unloaded NGFfrom NGF NPs. To determine the fractions containing NGF, 200 μl of NGFsolution (10 μg/ml) were added on a Sepharose 4B-CL column and elutedwith PBS. Twelve fractions (about 1 ml for each) were collected andmeasured for the concentrations of NGF using a Sandwich ELISA methoddeveloped based on a Sandwich ELISA kit for NGF (R&D System,Minneapolis, Minn.). In a separate experiment, 200 μl of NGFHDL-mimicking NPs were eluted from the same column. The intensity ineach fraction was measured using a Delsa Nano HC particle analyzer(Beckman Coulter, Calif.) to determine fractions containing NPs. Theconcentrations of NGF in fraction 5 to fraction 10 were measured andadded together to calculate the amount of unloaded NGF. Loading andentrapment efficiency of NGF were calculated using equation (1) and (2)as described above.

Statistical analysis of the data including ANOVA and t-test, whereverneeded, was conducted using Graph Pad Prism software. Results wereconsidered significant if p<0.05.

Results

Optimal procedure for nanoparticle preparation. Significant efforts havebeen devoted to the use of recombinant lipoprotein-like NPs as drugdelivery vehicles and diagnostic agents, because most of these particlesresemble natural lipoprotein structures and are considered highlybiocompatible and safe. Given the limitations of currently availablepreparation methods for scale-up of HDL-mimicking NPs, the inventorstested five different procedures to prepare the HDL-mimicking NPs byself-assembly. Table 1 below shows the results for four procedures andFIG. 2 shows the detailed study for the procedure using water at RT with5-min homogenization. Efficient mixing is the key to prepare the NPsless than 200 nm. Increase of temperature did not help decrease ofparticle size. Since homogenization is a common technique used toprepare liquid formulations in industrial scales, the inventors enhancedthe mixing efficiency by homogenization. With short-time homogenization,the inventors produced particle size at 183.9 nm with a narrow sizedistribution (P.I.<0.3). To further evaluate the influence ofhomogenization time on particle size, different homogenization time wasstudied. As shown in FIG. 2, there were no significant differences inparticle size among 3-min 4-min, 5-min and 6-min homogenization(p>0.05). Thus, 5-min homogenization was selected to prepare NPs. Thenew preparation method developed here is easy to be scaled up withappropriate reproducibility.

TABLE 1 Evaluation of preparation procedures for blank HDL-mimickingnanoparticle formation. Particle Prepaation conditions size (nm)^(a)P.I.^(b) 50° C. water + 30 min stirring at 50° C. 347.7 ± 19.4 0.322 ±0.0075 50° C. water + 30 min stirring at RT  297.7 ± 21. 5 0.296 ±0.0118 RT water + 30 min stirring at RT 335.4 ± 18.7 0.320 ± 0.0125 50°C. water + 5 min homogenization 183.9 ± 7.0  0.276 ± 0.030  (^(a)Thedata are presented as the mean of the mean particle size of NPs indifferent batches ± SD (n = 3); ^(b)P.I. means polydispersity index thatindicates size distribution of NPs. When P.I. < 0.35, NPs present as onesingle peak in the measurement (n = 3).)

Prototype HDL-mimicking NPs by Taguchi array. Accurate amounts ofexcipients in the NPs are keys to prepare self-assembled NPs. Asmentioned above, natural HDLs are composed of multiple components.Experimental design based on a statistical method is desired tofacilitate the finding of the accurate composition of the NPs formed byself-assembly. The inventors have used Taguchi array combined withsimplex optimization to develop paclitaxel NPs in a previous study (Donget at., 2009). Taguchi array effectively directed the nanoparticledevelopment and optimization. Hence, the inventors chose Taguchi arrayto develop and optimize the HDL-mimicking NPs in this study. Thedetailed rationale to design the Taguchi array is described in theMethod section. The results show in Tables 2A and 2B. Without TPGS,particle size was >250 nm (Tables 2A and 2C). The addition of TPGSdecreased particle size (<200 nm) and also narrowed size distribution(Tables 2B and 2D), but further increasing TPGS did not influenceparticle size as compared the batch 2-1 to other batches. The ratio ofphospholipids and CO did not influence to particle size as smallparticle size (<200 nm) was obtained in both Taguchi arrays (Tables 2Band 2D) As shown in Table 2B, batch 2-1, 2-4, 2-6 and 2-7 gave smallerparticle size compared to other batches. However, the total amount ofthe surfactants in batch 2-1 was very high, potentially leading toinstability of NPs; thus, batch 2-4, 2-6 and 2-7 were selected to loadApo A-I. In Table 2D, all four batches produced similar NPs. Theinventors chose batch 4-2 and 4-3 to represent batches with differentamounts of CO to load Apo A-I.

Apo A-I entrapment efficiency. The inventors used membrane separation tomeasure EE % of Apo A-I. Proteins have trend to bind with separationmembranes. Thus, the inventors measured the recovery of Apo A-I fromAmicon Ultra. The result showed that about 50% Apo A-I were detected inthe filtrate after the initial centrifugation. After the inventors used400 μl of water to wash the membrane, the recovery of Apo A-I in thefiltrate was 84.3%±4.5, demonstrating that the method was sufficient tocollect free Apo A-I in the filtrate. The inventors loaded Apo A-I intothe batches highlighted in Table 2B and 2D in order to prepareHDL-mimicking NPs. Different conditions were tested to load Apo A-I intothe NPs. The results showed that the initial 4-hour stirring at RT wascrucial to get homogenous NPs, and incubation overnight was important toget appropriate EE % of Apo A-I. Thus, the inventors selected 4-hourstirring at RT followed with incubation at 4° C. overnight to load ApoA-I. It was observed that drug formulations with TPGS resulted in highdrug encapsulation efficiency along with high cellular uptake andtherapeutic effects in in vitro and in vivo respectively (Zhang et al.,2012). To understand the influence of TPGS on EE % of Apo A-I, theinventors also selected batch 1-6 from Table 2A and batch 3-3 from Table2C as representative batches to load. Apo A-I. As shown in Table 3, allof the batches (batch 5-2, 5-3, 5-4, 5-6, and 5-7) that contained TPGSin the compositions had higher EE % of Apo A-I, compared to the batcheswithout TPGS (batch 5-1 and 5-5). These results suggested that additionof TPGS improved EE % of Apo A-I. The highest EE % of Apo A-I wasprovided by the batch 5-6. To clearly know the influence of Apo A-Iloading on its EE %, the inventors designed another two batches by onlyreplacing the amount of PC with Apo A-I while keeping the same amountsof other excipients in the batch 5-6 (Table 4). By this design, theinventors minimized the influence from the change of NP composition. Theprofiles show that increasing Apo A-I loading did not change EE % of ApoA-I (FIG. 3). The EE % of Apo A-I was over 26%—about 3-fold higher thanthose reported in literatures. Consequently, the real content of Apo A-Iin the NPs were over 16% close to the Apo A-I content in natural HDLs.The inventors chose the composition of the batch 5-8 to prepareNGF-loaded NPs. In the preparation, the inventors added about 0.14 mg/mlof Apo A-I to achieve a sufficient Apo A-I content in the NPs, whichdramatically decreased the use of Apo A-I compared to previouslyreported NPs.

Ion-pair complex for NGF. NGF is a 120-amino acid polypeptide homodimer.It presents as monomer with 13 KD and forms dimer by a disulfide bond inaqueous condition. Positively charged amino acids are dominate in theNGF monomer chain; however, after folding, the surface potential of theNGF dimer is negative as positively charged basic groups forms apositive groove at one end of the dimer that responsible for the bindingaffinity of NGF to its receptor. Therefore, the inventors hypothesizedthat a cationic polymer would be a suitable complex agent to form anion-pair complex with NGF to facilitate encapsulation of NGF into theNPs. First, the inventors tested if cationic polymers could formcomplexes with NGF. After mixing protamine with NGF at 1:1 ratio (w/w),the inventors easily visualized formation of white precipitates,directly indicating the formation of the complex. Next, the inventorsmeasured particle size and zeta potential of the complexes that wereformed by mixing each cationic polymer with NGF at different ratios. Asexpected, zeta potential changed from positive to negative whiledecreasing the concentrations of protamine, protamine sulfate USP andpoly-D-lysine (Table 5 below). The results confirm that NGF has negativecharge on the surface and using cationic polymers for complexation isappropriate for NGF. PC and SM are neutral phospholipids and TPGS is anon-ionic surfactant. PS is negative-charged phospholipids. Thus, thewhole NPs are negatively charged. A desirable complex should not onlycontain a minimal amount of the cationic polymer to produce sufficientcomplexation but also keep the complex slightly positively charged to beentrapped into the negatively charged HDL-mimicking NPs. As shown inTable 5, large aggregation was shown at the ratio of 1:1 of NGF toprotamine, suggesting that a complex formed and tended to aggregate.Importantly, at the ratio of 1:1, the complex had slightly positivecharge, which was preferred as described above. Compared with othertested protamines, protamine sulfate USP showed more favorite propertiesin terms of particle size and zeta potential. Moreover, protaminesulfate USP is approved by the Food and Drug Administration forinjection. Therefore, the inventors chose protamine sulfate USP as theion-pair agent to prepare NGF HDL-mimicking NPs. In contrast toprotamine, the inventors did not observe the same trend on particle sizefor poly-D-lysine. The ratio of NGF to poly-D-lysine at 1:1 and 1.2:1produced similar particle size, but the zeta potential was moresensitive for the change compared to protamines. These results suggestedthat protamines were superior to poly-D-lysine for NGF as thecomplexation using poly-D-lysine could more difficult to be qualifiedand controlled than those using protamines. The inventors includedpoly-D-lysine in the following study as a comparison. One concern whileusing charge-charge interaction for formulations is instability of theion-pair complex because of the competition from other ions inphysiological fluid. To verify the stability of the NGF complexes, theinventors mixed the NGF/protamine USP or NGF/poly-D-lysine complexeswith PBS and then measured particle size. In PBS, particle size of theNGF/protamine complex and the NGF/poly-D-lysine complex was 725.3 nm and957.6 nm, respectively, indicating both complexes were stable.

TABLE 5 Ion-pair complexes of protamines or poly-D-Lysine with NGF atdifferent ratios. Protamine Protamine Protamine Poly- free base saltfrom salmon sulfate USP D-Lysine NGF:Polycation Size Potential SizePotential Size Potential Size Potential (w/w) in water (nm) (mV) (nm)(mV) (nm) (mV) (nm) (mV) 0.8:1 554.3 12.22 278.5 0.34 562.6 0.86 529.30.54  1:1 863.6 0.58 589.4 0.22 802.6 0.30 805.5 −0.82 1.2:1 596.4 −0.71543.7 0.59 356.0 −0.32 830.0 −4.53

NGF-loaded HDL-mimicking NPs. To load 10 μg/ml of NGF, the inventorsmodified the composition of the batch 5-8 (Table 4) by increasing eachexcipient for 1.5 times. The final composition of the NGF HDL-mimickingNPs is shown in Table 6. The NGF loading was 3.2% and the Apo A-Iloading was 50.8%. Two procedures to add the NGF complex into the NPswere evaluated. In both procedures, adding NGF complex before and afterhomogenization, did not show difference on particle size and sizedistribution. To protect the bioactivity of NGF after nanoparticlepreparation, the inventors decided to add NGF complex afterhomogenization. Also, after addition of Apo A-I, stirring the NPs at RTovernight provided higher EE % of NGF compared to incubation at 4° C.overnight. As a consequence, NGF HDL-mimicking NPs were prepared bystirring at RT overnight after adding Apo A-I.

To measure the EE % of NU, the inventors first tried to use Amicon Ultra(molecule cutoff 100 kDa) to separate free NGF and NGF-loaded NPs.However, free NGF did not pass the membrane, probably due to theformation of the high molecular weight of the NGF dimer (26 KDa) inaqueous solution. The inventors next tested several gel filtrationcolumn including Sephadex G-50, Sephadex G-100, Sephacryl S-100 andSepharose CL-4B. Finally, Separhose CL-4B completely separated NGF NPsand free NGF. As shown in FIG. 4, fractions of 2 to 4 contained NGF NPs.The inventors calculated the EE % of NGF based on the concentrations offree NGF from fraction 6 to fraction 10 after the column separation.Different ELISA methods were evaluated to quantitatively measure theconcentration of NGF. A direct ELISA method worked very well for NGFstandard solution that was in PBS. However, cationic polymers, protaminesulfate USP and poly-D-lysine, increased the NGF absorbance in thedirect ELISA method. Next, the inventors evaluate a commercial NGF ELISAkit. Protamine sulfate USP and poly-D-lysine did not interfere with NGFmeasurement using the sandwich ELISA method. Characterization of NGFHDL-mimicking NPs is shown in Table 7 below. Both NGF HDL-mimicking NPshad relatively narrow size distribution. D90, the size which 90% of thedistribution lies below, was smaller than 550 nm and D10, the size which10% of the distribution lies below, was bigger than 75 nm. As expected,NGF/protamine sulfate USP NPs had higher NGF EE % than NGF/poly-D-lysineNPs. The variation of zeta potential on NGF/protamine sulfate USP NPsalso was smaller than that of NGF/poly-D-lysine NPs. It could be becausethe charge density of poly-p-lysine is relatively high compared toprotamine sulfate USP; thus, small change on poly-D-lysine amountssignificantly influenced complex formation and zeta potential. Also, theNGF/poly-D-lysine complex had negative zeta potential that may notprefer the negatively charged NPs. The final NGF NPs had negative zetapotential that is favorable for cell uptake and nanoparticle stability.Hazardess organic solvents (e.g. chloroform) were not used, and allexcipients in the NPs are naturally present, minimizing the toxicity ofthe NPs. In this study, PBS was used to wash the gel filtration columnto separate free NGF and NGF NPs for measurement of the EE %. UnloadedNGF and loosely bound NGF (on the nanoparticle surface) were separatedand washed out as free NGF from fraction 6 to 10. Therefore, the 65.9%of NGF measured for the EE % should be entrapped in the core of the NPsso that they did not dissociate from the NPs during the columnseparation and elution by PBS. This suggests that the HDL-mimickingα-tocopherol-coated NPs could protect NGF from degradation andsystemically deliver NGF to treat diseases.

TABLE 7 Characterization of the HDL-mimicking α-tocopherol- coated NGFNPs using protamine sulfate USP and Poly- D-Lysine as ion-pair agents,respectively (n = 3). NGF HDL- Particle EE % Zeta poten- mimicking NPssize (nm) P.I. of NGF tial (mV) Protamine 171.4 ± 5 0.289 ± 0.012 65.9 ±1.4 −12.5 ± 1.9 sulfate USP Poly-D-lysine  152 ± 5 0.265 ± 0.012 49.1 ±1.7 −24.9 ± 8.1

Physical stability studies of NPs. Stability measurement for optimizedHDL-mimicking NPs was performed on basis of particle size and. P.I. Thebatch 4-2 in Table 2D was stable over six months at 4° C. (FIG. 5).Batch 2-4, 2-6 and 2-7 in Table 2B was stable were stable over threemonths at 4° C. (FIG. 6). The prototype HDL-mimickingα-tocopherol-coated NPs (batch 5-8 in Table 4) were stable over twomonths at 4° C., and the NGF HDL-mimicking NPs were stable over onemonth at 4° C. However, considering degradation potentials of both ApoA-I and NGF during long-term storage in aqueous solutions, the inventorsare also studying the lyophilization of the NGF NPs to make them aspowders for long-term storage. The stability results demonstrated thatthe NPs developed in this study were stable with or without Apo A-I. Theinventors developed not only the NGF HDL-mimicking α-tocopherol-coatedNPs but also the stable lipid NPs that did not contain Apo A-I by usingTaguchi array. These lipid NPs will be further characterized andevaluated for their potential applications for drug delivery.

The inventors also demonstrated that docetaxel (an anti-cancer drug)could be encapsulated into the HDL-mimicking NPs. This indicates the NPsdescribed herein have potential to deliver not only small molecules butalso large molecules. Also, even without Apo A-I the inventors were ableto generate stable NPs. Therefore, the novel NPs can be considered aslipid NPs (without Apo A-I), but also as HDL-mimicking NPs that couldtake advantage of HDL NPs. These unique properties of the NPs developedin this invention will broaden their applications as drug deliverysystems to treat various diseases, such as the CNS disorders, cancersand eye diseases.

Prophetic Example 2 LR-Targeted NPs to Treat Docetaxel ResistantMetastatic Prostate Cancer

The inventors will evaluate the synergistic efficacy of prostate cancerspecific targeted nanoparticles (NPs) containing both docetaxel (DTX)and an antisense oligonucleotide (ASO) to overcome DTX resistance inmetastatic castration-resistant prostate cancer (mCRPC).

Novel NPs to Encapsulate Both DTX and OGX-011

The novel NP delivery system should incorporate DTX and ASO into one NP.As described in Example 1, the inventors have recently developed thenovel high-density lipoprotein (HDL)-mimicking NPs to encapsulate nervegrowth factor (NGF). The novel NPs have a narrow particle sizedistribution (polydispersity index, <0.3). Their particle size (<200 nm)is ideal to avoid hepatocytes uptake in liver (particles <100 nm) andsplenic filtration (particles 250 nm), but take the advantage of EPReffect (particles 100-200 nm) (Chen and Weiss, 1973 and Huang and Liu,2011). In this new formulation, NGF formed an ion-pair complex withprotamine and then the complex was encapsulated into the lipid core ofthe HDL NPs. Similar with NGF, OGX-011 will form an ion-pair complexwith a positively charged polymer to facilitate the entrapmentefficiency of OGX-011 in the NPs. Different from natural HDL, theinventors added TPGS into the NPs to further stabilize HDL-mimicking NPsand improve Apolipoprotein A-I (Apo A-I) entrapment efficiency.Interestingly, the inventors found that the novel NPs were stable atleast for 3 months at 4° C. even without Apo A-I. 10% DTX (w/w,drug/total excipients) with >75% entrapment efficiency were successfullyloaded into the novel NPs (without Apo A-I), DTX NPs significantlydecreased the IC₅₀ of DTX in DTX-resistant prostate cancer cellscompared to free DTX (FIG. 8), which proved the uptake of the NPs incancer cells.

R11 for Targeting Prostate Cancer and Gene Delivery

Additionally, pegylated R11-coated DTX-ASO NPs should provide aprolonged circulation of DTX and ASO compared with free DTX and freeASO. Encapsulation of ASO into the core of the NPs will prevent thedegradation of ASO in the blood. Coating PEG on NP surface will create ahighly solvated polymer layer at the NP surface, which causes a stericexclusion against the opsonin protein binding and consequently reducesthe reticuloendothelial system (RES) uptake. However, the amount of PEGcoated on NPs is critical for the steric exclusion (Huang and Liu,2011). Based on the preliminary data on pegylated PX BTM NPs, 10% of PEGmay be appropriate to provide a long circulation of NPs but also releasethe carried drug efficiently.

Despite widespread reports of in vitro and in vivo results with activelytargeting NPs, many studies finished with incomplete characterization ofthe NPs (Juliano et al., 2014). The amount of targeting ligands,physical and chemical properties, the impact of conjugation on ligandaffinity, and pharmacokinetics of the targeted NPs remain largelyuninvestigated. The information is important for reproducibility andapplication of active targeting strategies. Therefore, the inventorswill fully characterize Brij 700-R11 conjugate and R11-coated DTX-ASONPs.

Engineer Pegylated R11-Coated DTX-ASO NPs and Evaluate Them In Vitro

Proposed Methods'and Materials. The inventors will prepare andcharacterize pegylated R11-coated NPs. Following the promising resultsfrom Brij 700-TGF-α conjugation, the inventors will tresylate the —OHgroup of Brij 78, and then tresylated Brij 700 will react with theN-terminal amine group of R11. Briefly, Brij 700 will be dissolved indichloromethane and tresyl chloride and pyridine will be added to Brij700 solution by a drop-wise method at 0° C. The reaction solution willbe stirred for 18 hours at room temperature under N₂. Then, the organicsolvents will be removed by a rotary evaporator and the precipitateswill be purified by acidized ethanol. To prepare Brij 700-R11 conjugate,100:1 (molar ratio) of tresylated Brij 700 and R11 will be mixed anddissolved into 0.1 M HEPES buffer (pH 7.4). Brij 700-R11 conjugate willbe separated and purified using a Sephadex G-25 column. PAGE gel will beused to confirm the purity of the conjugate. The final concentration ofR11 in the purified Brij 700-R11 will be measured by Bradford assay.

The inventors will coat both Brij 700-R11 and DSPE-PEG-2000 on thesurface of the NPs. Briefly, phosphatidylcholine (PC), sphingomyelin(SM), phosphatidylserine (PS), cholesteryl oleate (CO), and TPGS will bedissolved in ethanol and mixed and dried under N₂. One milliliter ofwater will be added into the mixture. After 5-min homogenization, theinventors should get the NPs with particle size about 200 nm. And then,a mixture of Brij 700-R11 and DSPE-PEG-2000 will be added into the NPsand incubated at 30° C. for 15 min to coat the conjugate and PEG on thesurface of the NPs. The NPs will be put into Amicon Ultra (MW cutoff 100KDa) to separate free components and encapsulated ones. Free R₁₁ andDSPE-PEG-2000 in the filtrate will be measured using Bradford assay andHPLC with a refractive index detector, respectively, to determineentrapment efficiencies. The NPs will be labeled with BODIPY byincorporating cholesteryl BODIPY 542/563 C11 (Life Technology) into theNPs. Prostate cancer cell lines including DTX-resistant DU145, PC-3 KD1and C4-2 Neo will be treated with BODIP-loaded R11-coated NPs for 30min. The bioactivity of the pegylated R11-coated NPs will be determinedbased on the uptake of the fluorescence (BODIPY) in the cells.Additionally, the location of BODIP will be determined usingfluorescence microscopy to evaluate if BODIP presents in the cytosol.The ratio and amounts of Brij 700-R11 and DSPE-PEG-2000 will beoptimized to provide the optimal uptake and also about 10% ofDSPF-PEG-2000 on the NPs.

Next, the inventors will prepare and characterize pegylated R11-coatedDTX-ASO NPs. Different polyanions, such as polylysine and protamine,will be tested to form a suitable ion-pair complex with OGX-011.Briefly, OGX-011 will be mixed with a polyanion at different ratios.Particle size and zeta potential of the complexes will be measured. Theoptimal ratio will provide particles with zeta potential about 0,indicating the neutralization of the charge on OGX-011. The optimalcomplex will be added into the R11-coated NPs described above. Briefly,PC, SM, PS, CO and TPGS will be mixed and dried. The complex will beadded into the dried mixture and mixed for 20 min. After this, 1 mlwater will be added into the mixture and homogenized to form the NPs.And then, Brij 700-R11 will be added into the NPs as described above. Tomake pegylated NPs, a mixture of Brij 700-R11 and DSPE-PEG 2000 will beadded into the NPs.

Particle size, P.I. and zeta potential will be measured by Delsa Nano HC(Beckman Counter). A short-term physical stability will be evaluatedbased on particle size at 4° C. for 3 months. To measure the entrapmentefficiencies of DTX and OGX-011, free DTX and OGX-011 will be separatedfrom the NPs by centrifugation using Amicon Ultra (MW cutoff 100 KDa) at4° C. The free DTX in the filtrate will be measured by HPLC. FreeOGX-011 in the filtrate will be analyzed using a specially validatedELISA/cutting method that was used in Phase I study of OGX-011 (CTBRBio-Research Inc., Canada) (Chi et al., 2005). The in vitro release ofDTX and GU81 from pegylated R11-coated DTX-ASO NPs will be performedusing Amicon Ultran (MW cutoff 100 KDa). Briefly, 200 μl of the NPs willbe added into 20 ml PBS buffer and shake at 135 rpm over time at 37° C.At certain time intervals, released DTX and OGX-011 will be separatedfrom the NPs using Amicon Ultran and measured as described above. Inparallel, the sample will be taken to measure particle size to evaluatephysical stability of the NPs in PBS buffer at 37° C. To further mimicthe release in the blood circulation, the release study will be alsoconducted in the whole blood as reported previously (Feng et al., 2013).Briefly. The NPs will be mixed with the fresh mouse blood and incubatedfor 24 hours at 37° C. with shaking. At a certain time point, 250 μl ofblood will be withdrawn to get the plasma. A 15 cm Sepharose CL-4Bcolumn (GE Healthcare, US) will be used to separate released Brij78-R11, DTX and OGX-011. from the NPs. Free Brij 78-R11, DTX and OGX-011will pass through the column to determine which fractions contain theagents. The corresponding fractions will be collected to measure thereleased agents. Released Brij 78-R11 will be measured by HPLC asreported previously with modification (Miklan et al., 2009). ReleasedDTX will be measured using PX as an internal standard by an AgilentG6460 Triple Quad LC-MS/MS as described previously [30]. ReleasedOGX-011 will be determined by the ELISA/cutting method as describeabove.

The overall criteria for the final pegylated R11-coated DTX-ASO NPsinclude (1) particle size <200 nm, (2) P.I.<0.3 (monodispersed), (3)entrapment efficiency >80% with minimum drug concentrations of 150 μg/mlfor DTX and 100 μg/ml for GU81, (4) physical stability based on particlesize for one month at 4° C. and 24 hours at 37° C., and (5) less than50% release of Brij 78-R11, DTX and OGX-011 within 8 hours in PBS or theblood.

The amounts of DTX and OGX-011 in the NPs will be calculated based onanimal studies available in literatures and also the animal studies asdescribed below. It is critical that the targeting ligand can stay onthe NPs with the drug for a period of time to achieve tumoraccumulation. The in vitro release studies will test this property andassist further NP optimization. All related analytical methods eitherreported in literature or developed by the inventors will be utilized.If rapid releases are observed, the inventors will change the NPcomposition using the compositions generated from previous Taguchiarray. Also, the inventors may use different polyanions (i.e. hyaluronicacid) to condense OGX-011 and form a stable complex.

The inventors will also evaluate pegylated R11-coated DTX-ASO NPs inprostate cancer cells. They will use DTX-resistant DU145 (androgenreceptor [AR] negative) and several DAB2IP-knockdown prostate cancercell lines generated by Dr. Hsieh. DAB2IP is characterized as a potenttumor suppressor in prostate cancer progression and the loss of DAB2IPis associated with chemoresistance of mCRPC (Wu et al., 2013). TheseDAB2IP-knockdown (KD) cell lines showed significantly high resistancefor DTX, and also upregulated sCLU gene expression (Wu et al., 2013).Among these cell lines, PC-3 (AR negative) and C4-2 (AR positive) havebeen characterized to express LR and used to test R11 uptake.Specifically, six cell lines will be used for this project: DU145control cell line and resistant cell line, PC-3 control cell line andresistant cell line (KD1), and C4-2 control cell line (D2) and resistantcell line (Neo). OGX-011 will be fluorescently labeled with Cy3 toassist characterization of OGX-011 in prostate cancer cells. Todetermine the subcellular localization of OGX-011, cells will be treatedwith the NPs for 30 min. After fixation, cells will be counterstainedwith DAPI. The cellular distribution of Cy3-OGX-011 will be examinedunder fluorescence microscope. Cytotoxicity of the NPs will be measuredusing MTT assay at 72 hours and compared to controls including the emptypegylated R11-coated ASO NPs, the mixture of the empty NPs with DTX andASO, pegylated R11-coated DTX NPs and pegylated DTX NPs. Cell apoptosisafter treated with pegylated R11-coated DTX-ASO NPs will be tested usingin situ Cell Death Detection Kit POD (Roche Applied Science). sCLUexpression on the treated cells with OGX-011 and/or DTX will be assessedby Western blotting as reported previously (Sowery et al., 2008).

With well-controlled NP preparation in previous Tasks, the inventorsexpect that Cy3-OGX-011 will be located in the cytosol. OGX-011 willdecrease the gene expression of sCLU in the resistant cells. PegylatedR11-coated DTX-ASO NPs will show superior cytotoxicity compared to DTXNPs and free DTX in the resistant cells. All bioassay methods areavailable for the studies and the inventors do not expect the problemson them. If OGX-011 does not present in the cytosol, instead of coatingPEG and R11 on the NPs, the inventors will coat Apo A-I on the NPs tomake HDL-mimicking NPs. HDL provide significant opportunities as genedelivery vehicles because they are endogenous carriers of miRNAs. Datahas previously demonstrated that reconstituted HDL NPs escaped endosomeand facilitated high efficient systemic delivery of siRNA in vivo(Shahzad et al., 2011 and McMahon et al., 2014), Since HDLs are naturalNPs in the body, HDL-mimicking NPs have potential to escape the RES,leading to a long circulation in the blood. Scavenger receptor type B-I(SR-BI) is responsible for natural HDL uptake. Among the normal tissues,only liver has high SR-BI expression, whereas others have minimal to noexpression (Shahzad et al., 2011). However, SR-BI overexpresses incancer cells. Thus, DTX-ASO HDL-mimicking NPs will still have activelytargeting effect in cancer cells.

Evaluate Pegylated R11-Coated DTX-ASO NPs In Vivo

For animal studies, all samples will be sterilely prepared in 10%lactose to make them isotonic. A unique feature for the NPs is thatconcentrated NPs can be made by increasing the amount of each componentin the NPs at least 20 times. The maximal tolerate dose of single doseof DTX in mice was reported as 15-33 mg/kg (Dykes et al., 1995). Tenmg/kg of OGX-011 was a safe dose for mice (Sowery et al., 2008). Thus,the inventors will select three doses for subcutaneous (s.c.) modelsbased on in vitro IC50s to fit the range of 3-10 mg/kg of DTX andOGX-011. To meet the dose requirement, concentrated NPs will be preparedto allow 100 μl of i.v. injection in mice.

The inventors will first evaluate pharmacokinetics (PK) of pegylatedR11-coated DTX-ASO NPs in mice. PK studies will be performed in BALB/cmice. Five male BALB/c mice (4-6 weeks of age) will receive i.v.injection for each treatment through the tail vein. The inventors willtreat mice with 100 μl of pegylated R11-coated DTX-ASO NPs (1 mg/ml ofdocetaxel and 1 mg/ml of OGX-011) to give a dose of 5 mg/kg for bothagents. At given time intervals (0,1, 2, 3, 4, 6, 9 and 24 hours), micewill be sacrificed for blood and tissue collection. The plasma will bedivided to two portions to measure DTX and OGX-011 separately.Measurement of DTX in the plasma will be conducted by a LC-MS/MS asreported previously (Kim et al., 2013). OGX-011 will be directlyquantified from the plasma using the ELISA/cutting method as mentionedin Task 1.2. The PK parameters will be calculated with standardnoncompartmental analyses using Phoenix WinNonlin version 6.3 (Certara,St. Louis, Mo.). The C_(max), I_(max), T_(1/2), CL, AUC_(last), andAUC_(0-∞) will be calculated and compared to the controls including DTX,OGX-011, and R11-coated DTX-ASO NPs.

The inventors expect a prolonged circulation of DTX and OGX-011 by usingpegylated R11-coated DTX-ASO NPs. The initial loading of PEG will be10%; however, the inventors will optimize the PEG loading based on thePK results.

Next, the inventors will perform in vivo anti-cancer efficacy,biodistribution and toxicity studies. A s.c. model will be used fordose-finding experiments. An effective dose required to producesynergistic effect of DTX and OGX-011 in vivo will be determined byinjecting three doses of pegylated R11-coated DTX-ASO NPs (3, 5, and 10mg/kg of DTX and OGX-011) in prostate cancer bearing mice (n=8) (SeeVertebrate Animals). Mice will be treated once a week for three weekswhen the tumor volume reaches 50 mm³. Tumor size and mice will beweighted every three days. At the end of the study, mice will besacrificed and tumor, kidney, lung, heart, liver and spleen will beflash-frozen in liquid nitrogen. One third of tissues will be fixed forroutine histological examination to evaluate the toxicity. One third oftumors will be used to study for tumor immunohistochemical staining asdescribed previously (Sowery et al., 2008). The rest of tumors andtissues will be sonicated in RIPA buffer with a protease inhibitor. Thetotal cell lysate will be use to assess clusterin expression in tumorsby Western blotting, OGX-011 concentration in tumors and tissues by theELISA/cutting method and DTX concentration in tumors and tissues by aLC-MS/MS as described above. Saline, pegylated R11-coated NPs andpegylated DTX-ASO NPs will be used as controls.

To make the proposed studies relevant to the clinic setting, therapeuticefficacy of the NPs will be evaluate in bone metastatic models in SCIDmice. Since mCRPC can be both AR positive and AR negative, the inventorswill use PC-3 KD1 (or DTX-resistant DU145; AR negative) and C4-2 Neocells (AR positive) to establish the bone metastatic models. Thedetailed procedures on animal models are described in the section ofVertebrate Animals. Mice will be injected with the optimal dose selectedfrom Task 2.2 above. Total 7 treatment groups (See Vertebrate Animals)include saline, Texotere, empty NPs, pegylated R11-coated DTX NPs,pegylated DTX-ASO NPs, pegylated R11-coated DTX-ASO NPs, and a mixtureof pegylated R11-coated DTX NPs and ASO. To trace tumor growth, theinventors will monitor serum PSA levels (for AR positive tumor) and MRIto observe any delay of relapse. The inventors will harvest tumorbiopsies starting the end of last treatment and every two weeks forhistologic examination. The inventors will also determine the activityof bone stromal cell using Von Kossa staining, immunostaining forosteopontin or X-ray for osteoblastosis. In this study, the inventorswill also document the PSA-free survival based on the recurrent time ofPSA and the survival rate (Kaplan-Meier curve) based on the time ofanimal sacrifice (i.e., BLI intensity)

The inventors will decide which cell, PC-3 KD or DTX-resistant DU145,will be used for the metastatic model based on the outcome of otherstudies. Enhanced synergistic efficacy is expected from pegylatedR11-coated DTX-ASO NPs compared to the controls, especially the mixtureof pegylated R11-coated DTX NPs and OGX-011. sCLU expression in thegroup of pegylated R11-coated DTX-ASO NPs will be lower compared to thecontrols. Also, the inventors expect the actively targetingoutcome—enhanced accumulation in tumor by R11-coated NPs compared withuncoated NPs. If the proposed bioassay methods are not sensitive enough,the inventors will use radio-labeled DTX and OGX-011 for thebiodistribution study. Except of Brij 700, the components in the NPs areFDA-approved excipients and naturally exit in human body; thus, toxicityis not expected from the empty NPs. Since OGX-011 will be co-deliveredwith DTX by i.v. injection in the NPs, the inventors expect that a lowdose of OGX-011 may generate the synergistic effect. All tumor models inthis example have established in prior studies. The s.c. model is usednot only for dose finding but also for NP development. If s.c. modeldoes not yield results, the inventors will further optimize the NPsusing the strategies as described above and repeat the animal studies.

Example 3 Additional Applications of Nanoparticles

Small Molecules

Docetaxel (DTX) was dissolved in ethanol at 200 kg/ml.Phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylserine (PS),cholesteryl oleate (CO) and D-α-Tocopheryl polyethylene glycol succinate(TPGS) were dissolved in ethanol to prepare stock solutions at 1 mg/ml,respectively. The, 59 μl PC, 11 μl SM, 4 μl PS, 15 μl CO, 45 μl TPGS and75 μl DTX were added into a glass vial. After mixing, the ethanol isremoved under a gentle nitrogen stream. The mixture was homogenized at8600 rpm for 5 min at room temperature to form DTX NPs. DTX NPs werecharacterized by measuring particle size, size distribution, entrapmentefficiency.

DTX-resistant castration-resistant prostate cancer cells (DU145 cells)were treated with different concentrations of free DTX and DTX NPs,respectively. After 72 hours, MTT assay was used to measure cellviability and the IC₅₀s of free DTX and DTX NPs were calculated.

The inventors successfully loaded 10% docetaxel (DTX) (w/w, drug/totalexcipients) with >75% entrapment efficiency into the novel NPs (withoutApo A-I). Particle size (˜170 nm) of DTX NPs and size distribution weresimilar with the original NPs. According to the cytotoxicity studies,DTX NPs significantly decreased the IC₅₀ of DIN in DTX-resistant CRPCcells compared to free DTX (FIG. 7), which also proved the uptake of theNPs in cancer cells.

Proteins

Materials and Cell Culture. Protamine from salmon, protamine grade X,protamine sodium salt USP, poly-lysine and cholesteryl oleate (CO),Sodium chloride, sodium acetate, Triton X-100, bovine serum albumin(BSA), phosphate buffer saline (PBS), phenylmethylsulfonyl fluoride(PMSF) and benzethonium chloride were purchased from Sigma (St. Louis,Mo.), Sephadex G-50, Sephadex G-100, Sephacryl S-100 and Sepharose CL-4Bwere also purchased from Sigma-Aldrich (St. Louis, Mo.). PC, SM, andphosphatidylserine (PS) were purchased from Avanti polar lipids(Alabaster, Ala.). TPGS was provided by BASF as a gift. Apo A-I waspurchased from Athens research and technology (Athens, Ga.). Recombinanthuman NGF was purchased from Creative Biomart (Shirley, N.Y.). Bradfordreagent was obtained from Thermo Scientific (Rockford, Ill.). Amiconultra centrifugal filters (0.5 mL) were obtained from Merck Millipore(Germany). Float-A-Lyzer G2 Dialysis device (MWCO 300 kDa) was purchasedfrom Spectrum Laboratories (Rancho Dominguez, Calif.). Human beta-NGFDuoSet ELISA kit was purchased from R&D Systems (Minneapolis, Minn.).

Animals. Bcl mice (adult males, 25˜30 g) were purchased from CharlesRiver Laboratories (Wilmington, Mass.). All animal experiments werecarried out under an approved protocol by the Institutional Animal Careand Use Committee at the University of North Texas Health ScienceCenter.

Optimization of preparation procedure for prototype HDL-mimicking NPs.Blank HDL-mimicking NPs were prepared by a self-assembly method. Tomaintain NGF bioactivity after NP preparation, we chose low temperature(50° C.) or room temperature for preparation. All excipients weredissolved in ethanol to prepare stock solutions. PC (43.1%), SM (8.1%),PS (2.7%), CO (7.7%) and TPGS (38.4%) (percentages based on w/w) wereadded into a glass vial to form a thin film after removing ethanol bynitrogen. And then 1 ml of milliq water was added into the vial. Fivedifferent procedures were evaluated to hydrate the film to form NPs,including: 1) adding water at 50° C. and stirring at 50° C. for 30 minat 600 rpm, 2) adding water at 50° C.; and stirring at room temperature(RT) for 30 min at 600 rpm, 3) adding water at RT and stirring at RT for30 min at 600 rpm, and 4) adding water at 50° C. and homogenizing 5 minusing a homogenizer at 8600 rpm, and 5) adding water at RT andhomogenizing 5 min using a homogenizer at 8600 rpm. To further evaluatethe influence of homogenization time on NP formation, the mixtures werehomogenized for 0, 1, 2, 3, 4, 5, and 6 min after adding water at RT.After preparation, particle size and polydispersity index (P.I.) of NPswere measured using a Delsa Nano HC particle analyzer (Beckman Coulter,Calif.) at 90° light scattering at 25° C.

Development of prototype HDL-mimicking NPs. Nanoparticles without ApoA-I. PC, SM and PS were selected as phospholipid components and CO wasselected as the lipid component to develop the HDL-mimicking NPs. Tosimplify the design and quickly find the optimal compositions, weconsidered phospholipids as one variable that include PC, SM and PS. Thepercentage of each phospholipid in the total phospholipids excluding COwas fixed as PC (76%), SM (14%) and PS (10%), which is close to thecomposition of phospholipids, but doubled the amount of PS, compared tothe composition of natural HDLs. To evaluate different ratios ofphospholipids and CO, the inventors designed two arrays. In the array #1(Table 2A and 2B, above), the ratios of total phospholipids and CO werecontrolled in a range of 0.6 to 1.6 (total phospholipids/CO, w/w). Thisarray for 3 levels 2 variables (phospholipids and CO) was used to givethree different concentrations for each excipient. The array #2 (fable2C and 2D, above), an array for 2 levels 2 variables, was used to givethe different ratios of total phospholipids and CO in the range of 4.9to 14 (total phospholipids/CO, w/w). In the array #2, the percentage ofeach phospholipid in the total phospholipids excluding CO was fixed asPC (80%), SM (15%) and PS (5%). NPs were prepared as described above.After forming the thin film, 1 ml of milliq water at RT was added intothe vial and homogenized for 5 min at 8600 rpm to form NPs. To makeTPGS-coated NPs, certain amounts of TPGS were added into thecompositions in Tables 2B and 2D to give a total surfactant(phospholipids+TPGS) in a range of 60 μg/ml to 120 μg/ml. Particle sizeand P.I. were measured as described above.

Optimization of loading Apo A-I in the prototype HDL-mimicking NPs.Based on the particle size and size distribution, the optimalcompositions were selected to load Apo A-I, which are highlighted inTables 2A-D. After homogenization for 5 min as described above, acertain amount of Apo A-I was added into each composition (Table 3,above). Four different conditions, including 2-hour stirring at RT,4-hour stirring at RT, 4-hour stirring at RT followed with incubation at4° C. overnight, and 4-hour stirring at RT followed with stirring at 4°C. overnight, were evaluated to load Apo A-I. Particle size and sizedistribution were measured as described above. EE of Apo A-I wasanalyzed by ultrafiltration. Briefly, 0.2 ml of the NPs were added intoAmicon Ultra (Molecular cutoff 100 KDa) and centrifuged at 14000 rpm at4° C. for 3 min. After this, 400 μl water were added into the insert ofAmicon to wash the membrane with the same centrifugation condition. ApoA-I was passed through the membrane and washed with the same approach asdescribed above to measure the recovery of Apo A-I in this separationmethod. The concentration of unloaded (free) Apo A-I in the filtrate wasmeasured by Bradford assay, Loading and EE of Apo A-I were calculated asfollows:

% loading=(drug added into NP)/(total weight of excipients+drug)×100%  Eq. (1)

% EE=(1−unloaded drug/total drug added into NP)×100%   Eq. (2)

Further optimization on Apo A-I loading was studied based on thecomposition of the batch 4-2. To optimize Apo A-I loading, differentamounts of Apo A-I were added into the NPs (Table 4, above) by changingthe amount of PC, but keeping the same amounts of SM, PS, CO and TPGS inthe batch 4-2. Loading and EE of Apo A-I were measured and calculated asdescribed above.

Particle size stability of prototype HDL-mimicking NPs at 4° C. Thephysical stability of the prototype HDL-mimicking NPs was assessed overtime at 4° C. Prior to particle size measurement, NPs were allowed toequilibrate to RT. One milliliter of NPs was used to measure theparticle size and P.I as described above.

Development of NGF-loaded HDL-mimicking NPs/Optimization of ion-paircomplex for NGF. To efficiently load NGF into the NPs, poly-lysine andthree types of protamines were tested to form an ion-pair complex withNGF. Protamines included protamine from salmon, protamine grade X andprotamine sodium salt USP. Poly-lysine, protamines and NGF weredissolved in water at the concentration of 1 mg/ml. NGF was added intopoly-lysine or protamine solutions at 0.8:1, 1:1, and 1:1.2 ratios(NGF:polymer, w/w). The complex was allowed to stand at RT for 10 min,and then diluted with 1 ml of water or PBS to measure particle size asdescribed above and also to measure zeta potential using the particleanalyzer. The optimal ratio of the complex was determined according toparticle size and zeta potential.

Preparation of NGF-loaded HDL-mimicking NPs. Poly-lysine and protamineUSP were selected to prepare NGF-loaded NPs. Briefly, 10 μg of NGF wasmixed with 10 μg poly-lysine or protamine USP (1:1, NGF:polymer, w/w)and kept for 10 min at RT to form the complex. PC, SM, PS, CO and TPGSethanol solutions (Table 6; above) were mixed and then ethanol wasremoved by nitrogen to form the thin film as described above. Twoprocedures were tested to add the NGF complex into NPs. In the firstprocedure, the NGF complex was added into the thin film, and then 1 mlof water at RT was added and homogenized for 5 min to incorporate NGF.In the second procedure, 1 ml of water at RT was first added into thethin film and homogenized for 5 min; and then the NGF complex was addedinto the solution. After the addition of NGF complex, the solution wasincubated at 37° C. for 30 min, and then stirred at RT for 30 min untilcooling in order to incorporate NGF. The defined amount of Apo A-I wasadded into each solution and stirred at RT overnight to form the finalNGF-loaded HDL-mimicking α-tocopherol-coated NPs. Particle size and zetapotential were measured as described above.

Determination of NGF entrapment efficiency in NGF-loaded IDOL-mimickingNPs. Gel filtration chromatography was used to separate unloaded NGFfrom NGF NPs. To determine the fractions containing NGF, 200 μl of NGFsolution (10 μg/ml) were added on a Sepharose 4B-CL column and elutedwith PBS. Twelve fractions (about 1 ml for each) were collected andmeasured for the concentrations of NGF using a Sandwich ELISA methoddeveloped based on a Sandwich ELISA kit for NGF. In a separateexperiment, 200 μl of NGF HDL-mimicking NPs were eluted from the samecolumn. The intensity in each fraction was measured using the particleanalyzer to determine fractions containing NPs. The concentrations ofNGF in fraction 5 to fraction 10 were measured and added together tocalculate the amount of unloaded NGF. Loading and EE of NGF werecalculated using equation (1) and (2) as described above.

In vitro release study. The release of NGF from NGF NPs (n=4) wasstudied using a dialysis method. The release medium was PBS (pH 7)containing 5% BSA to mimic the physiological condition in blood.Briefly, 200 μl NGF NPs and 400 l release medium were loaded into thedialysis tube (invco 300 kDa). Then the dialysis tube was placed into a30 ml release medium and shaken at a 37° C. at 135 rpm. At the timeintervals (1, 2, 4, 6, 8, 24, 48 and 72 hours), 100 μl of the releasemedium were withdrawn and replaced with an equal volume of fresh medium.The amounts of released NGF in the medium were analyzed by a NGFSandwich ELISA kit. As a control, free NGF (n=4) was studied inparallel.

Tissue distribution of NGF NPs. Mice were randomly divided to threegroups (n=3). Saline, free NGF and NGF NPs were injected, respectively,through tail vein at a dose of 40 μg/kg for each group. After injection,mice were sacrificed at 30 min, and blood, brain, liver, spleen andkidney were collected. Blood samples were centrifuged at 3400 rpm at 4°C. for 5 min to obtain plasma. Plasma and tissues were stored at −80° C.until analyzed. For tissue samples, 100 mg of tissues were suspended ina 10-times volume of extraction buffer (0.05M sodium acetate, 1.0 Msodium chloride, 1% Triton X-100, 1% BSA, 0.2 mM PMSF, and 0.2 mMbenzethonium chloride) and homogenized at 4° C. The concentrations ofNGF in plasma and tissues were measured by the Sandwich ELISA kit.

Statistical Analysis. Statistical analysis of the data including ANOVAand t-test, wherever needed, was performed using Graph Pad Prismsoftware. Results were considered significant if p<0.05.

Results—in vitro release study. The release profiles of free NGF and NGFNPs are shown in FIG. 14. Free NGF passed through the membrane readilyand reached 83% in the first hour. The inventors observed the tendencyof NGF to bind with the membrane when we tested the entrapmentefficiency. In the release studies, they added 5% BSA to reduce thebinding of NGF as well as matching the BSA concentration in blood. Theresult indicated that 5% BSA efficiently prevented the binding of NGF tothe membrane. With this advance, the inventors can accurately measurethe released NGF from NGF NPs. NGF NPs showed a slow release without aburst release. Only 5.5% of NGF was released within 1 hour. The releaseof NGF reached a plateau at 8 hours (9.9%) and kept over 72 hours. Therelease results demonstrated that NGF was entrapped in the core of theNPs, which aligns with the result of the entrapment efficiency.

Biodistribution. One of the inventors' hypotheses was that NPs canprotect NGF from degradation and control NGF release in order to improvethe half-life of NGF after intravenous injection. Hence, they measuredthe biodistribution of NGF NPs in mice. As shown in FIG. 8, NGF NPsincreased the plasma concentration of NGF by 1.7-fold compared to freeNGF. For tissues, NGF NPs decreased the tissue uptake by 3-fold inliver, 2.3-fold in kidney and 1.4-fold in spleen. The resultsdemonstrated that the NPs prolonged the circulation of NGF in blood. Asshown in the release studies (FIG. 14), NGF was entrapped inside the NPsand slowly released from the NPs. Thus, the NPs protected NGF fromdegradation in vivo, leading to a long circulation in blood and reduceduptake in tissues (FIG. 15). When the NPs are used to deliver NGF tobrain, the prolonged circulation would provide more opportunity for thebrain uptake compared to free NGF. Therefore, the novel HDL-mimickingNPs are very promising for delivery of NGF through intravenousinjection.

Neurite Outgrowth Study. It is important to maintain protein's activityafter the formulation of the NPs. Thus, the inventors chose to measurethe bioactivity of NGF HDL-mimicking NPs in PC12 cells for neuriteoutgrowth. They pre-coated a 6-well plate with rat tail collagen type I.They seeded PC12 cells at a density of 10000 cells/well to thepre-coated 6-well plate overnight to allow cells to attach on theplates. They diluted free NGF (10 μg/ml) and NGF HDL-mimicking NPs (10μg/ml) with the culture medium to prepare various concentrations at 0.5,1, 5, 10, 50, and 100 ng/ml using half-half dilution. Then, they added100 μl of sample into each well of the plate and culture for 4 days. Atday 4 they changed the medium to fresh medium containing thecorresponding treatment and then continue the treatment for another 3days. At day 7, they visualized cells by an inverted light microscopeand take the imaging from each well at random spots under 10×magnification.

FIGS. 13A-B represent the imaging of neurite outgrowth when the cellswere treated with 50 ng/ml of free NGF (FIG. 13A) and NGF HDL-mimickingNPs (FIG. 13B). When the treatment concentration was higher than 10ng/ml, neurite outgrowth was clearly observed by the microscope. Atthese high concentrations, free NGF and NGF HDL-mimicking NPs did notshow significant difference on the effect of neurite outgrowth. When theconcentration of NGF was lower than 10 ng/ml, neurite outgrowth cannotbe observed clearly for both free NGF and NGF HDL-mimicking NPs. Thus,the inventors have demonstrated the comparable bioactivity of NGFHDL-mimicking NPs with free NGF.

Micro RNA (Without Apo A-I)

The inventors utilized the novel NPs (without adding Apo A-I) toencapsulate microRNA-363 for prostate cancer. The preparation procedurewas similar with that of NGF HDL-mimicking NPs. Briefly,phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylserine (PS),cholesteryl oleate (CO) and D-α-Tocopheryl polyethylene glycol succinate(TPGS) were dissolved in ethanol to prepare stock solutions at 1 mg/ml,respectively. The, 59 μl PC, 11 μl SM, 4 μl PS, 15 μl CO, and 45 μl TPGSwere added into a glass vial. After mixing, the ethanol is removed undera gentle nitrogen stream. The mixture was homogenized at 8600 rpm for 5min at room temperature to form the prototype NPs. The inventors mixedmicroRNA-363 with protamine (1:2 ratio, w/w) to form the ion-paircomplex. Then, they added the complex into the prototype NPs andincubate them at 37° C. for 30 min. After cooling, they obtainedmicroRNA-363 loaded NPs.

The inventors used Cy5 labeled microRNA-363 to prepare the NPs andstudied the cellular uptake of the NPs by a confocal microscopy. Cellswere seeded in 12-well tissue culture plates at a density of 2×10⁴ cellsand incubated overnight at 37° C. Then the cells were treated with freemicroRNA-363 or microRNA-363 NP at 6.4 μg/ml for 3 hrs at 37° C. Thecells were washed with PBS, and then fixed with 4% formaldehyde. Thenuclei were stained with DAPI and the cells were mounted to glass slide.The red fluorescence of Cy5 was visualized with a confocal microscope.Cy5-labeled miRNA-363 was successfully encapsulated into the NPs. Theparticle size of microRNA-363 NPs was ˜170 nm with a narrow sizedistribution.

Moreover, the uptake study using confocal microscopy showed thatmiRNA-363 was located in the cytoplasm of PC3 and DU145 cells (FIG. 8).This result demonstrated that the inventors' novel NPs are promising toescape endosome and deliver miRNAs to cytoplasm. In addition, they canlyophilize the NPs without the loss of NP properties, which warrantslong-term stability of macromolecules and clinic translation. Therefore,the novel NPs have the ability to incorporate small molecules andmacromolecules. The inventors will use their novel NPs to deliver thecombination of small molecules and macromolecules, e.g., the combinationof microRNA-363 (or microRNA-145) and DTX.

Novel HDL-Mimicking TPGS-Coated NPs Delivering NGF, DTX and miRNA

To encapsulate NGF, the inventors used protamine or poly-D-lysine toform an ion-pair complex with NGF by charge-charge interaction, whichnormalized the surface charge of NGF to facilitate encapsulation of NGF.The characterization of novel NGF NPs is summarized in Table 7 (above).Note: phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylserine(PS), cholesteryl oleate (CO), vitamin E TPGS (TPGS).

In addition to appropriate particle size and entrapment efficiency, thezeta potential of NGF NPs is negative. Liposomes have been commonly usedfor gene delivery; however, safe and efficacious delivery in vivo israrely achieved due to toxicity, nonspecific uptake, and unwanted immuneresponse. The nonspecific response and toxicity are directly linked tothe positive charge on the surface of the liposomes necessary for thebinding of gene therapeutic agents. Thus, because of negative surfacecharge, the inventors' NPs will overcome the problems of liposomes.Importantly, novel NGF NPs had the same bioactivity compared to freeNGF, demonstrating that encapsulating of NGF into the NPs did not affectthe efficacy of NGF.

The inventors also successfully loaded 10% DTX (w/w, drug/totalexcipients) with >75% entrapment efficiency into the novel NPs (withoutApo A-I). DTX NPs significantly decreased the IC₅₀ of DTX inDTX-resistant CRPC cells compared to free DTX (FIG. 7), which proved theuptake of the NPs in cancer cells.

Novel HDL-mimicking TPGS-coated NPs delivering miRNA: Natural HDLs areendogenous carriers of miRNAs. Data demonstrated that reconstituted HDLNPs escaped endosome and facilitated high efficient systemic delivery ofsiRNA in vivo. The inventors developed their HDL-mimicking NPs based onthe composition of natural HDLs (Table 2B, above); and tested thefeasibility of their novel NPs to deliver miRNA-363. Similar with NGFNPs, the inventors used protamine to form the complex with miRNA-363 bycharge-charge action. Cy5-labeled miRNA-363 was successfullyencapsulated into the NPs. Moreover, the uptake study using confocalmicroscopy showed that miRNA-363 was located in the cytoplasm of PC3 andDU145 cells (FIG. 8). This result demonstrated that these novel NPs arepromising to escape endosome and deliver miRNAs to cytoplasm. Inaddition, the inventors can lyophilize the NPs without the loss of NPproperties, which warrants long-term stability of macromolecules andclinic translation. Therefore, the novel NPs have the ability toincorporate small molecules and macromolecules. These novel NPs can beused to deliver miRNA-145 as well as the combination of miRNA-145 andDTX.

SiRNA

Non-viral gene delivery systems, including lipid-based nanoparticles(NPs), polyethylenimine-based delivery system, dendrimers,poly(lactide-co-glycolide) NPs, have been extensively studied. Theinventors lipid-based NPs are novel in structure; they mostly like acombination of lipoplexes and HDL NPs. All components in these novel NPsnaturally exist and have no toxicity. Instead of using cationic lipidsthat caused the toxicity of lipoplexes, the inventors used protamine, aFDA-approved excipient, to form an ion-pair complex with macromolecules.By adding TPGS, the inventors were able to simply prepare the NPs by aself-assembly method, addressing the manufacturing difficulty and highcost of the NPs.

The preparation procedure was similar with that of microRNA-363 loadedNPs as described above. Briefly, phosphatidylcholine (PC), sphingomyelin(SM), phosphatidylserine (PS), cholesteryl oleate (CO) andD-α-Tocopheryl polyethylene glycol succinate (TPGS) were dissolved inethanol to prepare stock solutions at 1 mg/ml, respectively. The, 59 μlPC, 11 μl SM, 4 μl PS, 15 μl CO, and 45 μl TPGS were added into a glassvial. After mixing, the ethanol is removed under a gentle nitrogenstream. The mixture was homogenized at 8600 rpm for 5 min at roomtemperature to form the prototype NPs. The inventors mixed siRNA withprotamine (1:1 ratio, w/w) to form the ion-pair complex. Then, theinventors added the complex into the prototype NPs and incubate them at37° C. for 30 min. After cooling; they obtained siRNA loaded NPs.

To study the cellular uptake, the inventors used FITC-labeled modelsiRNA and Cy3-labeled anti-GAPDH siRNA to make the NPs and test them bya confocal microscope. Cells were seeded in 12-well tissue cultureplates at a density of 2×10⁴ cells and incubated overnight at 37° C.Then the cells were treated with free microRNA-363 or microRNA-363 NP at6.4 μg/ml for 3 hours at 37° C. The cells were washed with PBS, and thenfixed with 4% formaldehyde. The nuclei were stained with DAPI and thecells were mounted to glass slide. The green fluorescence of FTIC or theyellow fluorescence of Cy3 was visualized with a confocal microscope.

The inventors have encapsulated nerve growth factor (NGF) into the novelNPs. Over 65% of NGF was entrapped into the NPs with 170 nm of particlesize. Here, the inventors explored the novel NPs for encapsulation ofsiRNA. Both fluorescent-labeled siRNAs were successfully encapsulatedinto the NPs with over 75% entrapment efficiency. The particle size ofsiRNA NPs was ˜170 nm with a narrow size distribution. Cells treatedwith siRNA NPs showed internalization and accumulation of green (FTIC,FIG. 9) or yellow (Cy3, FIG. 10) fluorescence in cytosol. In contrast,no fluorescence was observed in cytosol of cells treated with free modelsiRNA and free anti-GAPDH siRNA. These results demonstrate that thenovel NPs are promising to escape endosome and deliver siRNA tocytoplasm for efficient gene transfection,

Use of Endosomal Escaping Agents to Further Modify of NP Composition

Endosomal escaping agents, also call fusogens, including MGDG(monogalactosyldiacylglycerol), diacylglycerol, polyphosphoinositidesand fatty acids (e.g., oleic acid and arachidonic acid), may beincorporated into the nanoparticle to enhance gene knockdown.

MGDG is a nonionic lipid and is a non-bilayer lipid; however, it plays acrucial role in membrane fusion. MGDG with conical morphology inducesnegative curvature, consequently forming inverted hexagonal phase (HII).Thus, MGDG has potential to break endosome membrane to assist genesescaping endosome, and thus incorporated MGDG in the inventors NPcomposition improve the efficiency of gene knockdown.

In addition, MGDG has a moiety of sugar (FIG. 11). Instead of using ApoA-I, the inventors included MGDG in the NP composition to prepareMGDG-coated NPs which could act as a “sugar” bead to target to GLUT1 (aglucose transporter in the blood-brain barrier) in order to facilitateacross the BBB.

MGDG, TPGS, DOPE and PC were dissolved in ethanol at 1 mg/ml,respectively. The excipients were mixed with certain amounts (Table 8).Ethanol was removed by nitrogen gas. The mixture was homogenized byusing a homogenizer at 8600 rpm for 5 min at room temperature to formthe prototype NPs. Alternatively, the mixture was sonicated for 1-5 minat room temperature using a sonication probe to form the prototype NPs.Then NGF or siRNA was formed the complex with protamine as describedabove. The complex was added into the prototype NPs and incubated for 30min at 37° C. The NPs were characterized for particle size, sizedistribution and entrapment efficiency.

To test the efficiency of gene knockdown, PC3-Luc+ cells, in which PC3cells (prostate cancer cells) were stably transfected with luciferase,were seeded in a 96-well tissue culture plate at a density of 8000cells/well and incubated overnight at 37° C. Nanoparticle was preparedbased on the batch compositions listed in Table 9 below. The procedureof preparation is described above. 20 μl of NPs were added to each wellwith 100 ul culture medium. The final siRNA concentration in each wellwas 12.3 pmole. After 48 h of the treatment, the medium was removed.Luciferase expression was measured by a luciferase assay. Proteins ineach well were measured by a BCA assay. Then, luciferase expression ineach well was normalized with protein concentration. The gene knockdownefficiency was represented by the percentage of luciferase/proteincomparing with the control (blank cells): % gene knockdown=Treatment(luciferase/protein)/Control (luciferase/protein)×100%

To evaluate the novel MGDG NPs to encapsulate NGF, the inventorsprepared NGF MGDG NPs. The compositions of novel NGF NPs and theircharacterization are shown in Table 8. The NPs had a narrow sizedistribution. For all batches in Table 8, the entrapment efficiency ofNGF or siRNA was over 95%.

To test the efficiency of gene knockdown, the inventors prepareddifferent MGDG NPs to encapsulate anti-luciferase siRNA (Table 9). Theresults of gene knockdown in PC3-KD1 Luc⁺ cells are shown in FIG. 12.The NPs composed of MGDG and TPGS shows a dose-dependent gene knockdownwhile changing the concentrations of MGDG. At the MGDG concentrations of25 μM (batch #2) and 50 μM (batch #1 and batch #4), the NPssignificantly decreased the expression of luciferase. Importantly, batch#1, batch #2 and batch #4 did not show significant difference comparedto the commercial gene transfection agent (lipofectamine) (#p>0.05),suggesting the great efficiency of the NPs for gene knockdown. Verylikely, MGDG induced membrane fusion to facilitate siRNA escapingendosome. According to the results, MGDG has better ability for genesilencing than DOPE. Therefore, the novel NPs in this invention havegreat potential for gene therapy.

TABLE 8 The compositions and characterization of the modified NGF NPscontaining MGDG PC TPGS MGDG NGF Protamine Particle Batch (μg) (μg) (μg)(μg) (μg) size P.I. 1 20 — 60 10 10 149.3 0.16 2 — — 60 10 10 252.30.063 3 10 — 60 10 10 352.3 0.211 4 — 10 60 10 10 286.7 0.141 5 — 20 6010 10 132.6 0.231

TABLE 9 The compositions and characterization of the modified siRNA NPscontaining MGDG MGDG TPGS PC DOPE siRNA Protamine Batch (μg) (μg) (μg)(μg) (μg) (μg) #1 240 80 — — 8 8 #2 120 200 — — 8 8 #3 25 295 — — 8 8 #4240 — 80 — 8 8 #5 120 — 200 — 8 8 #6 25 — 295 — 8 8 #7 — 80 — 240 8 8 #8— 200 — 120 8 8

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   U.S. Pat. No. 5,091,513-   U.S. Patent Application Publication No. 2005/0214860-   Aloe et al., J. Transl. Med., 10:239-5876-10-239, 2012.-   Apfel, Int. Rev. Neurobiol., 50:393-413, 2002.-   Balazs et al., J. Neurochem., 89:939-950, 2004.-   Barnett, et al., Exp. Neurol., 110:11-24, 1990-   Bruce, et al., Neurobiol. Aging, 10:89-94, 1989-   Chen and Weiss, Blood., 41:529-537, 1973.-   Chi et al., J. Natl. Cancer Inst., 97:1287-1296, 2005.-   Dong et al., Eur. J. Pharm. Biopharm., 72:9-17, 2009.-   Dykes et al., Invest. New Drugs., 13:1-11, 1995.-   Eriksdotter Jonhagen et al., Dement. Geriatr. Cogn. Disord.,    9:246-257, 1998.-   Feng et al., Adv. Healthc. Mater., 2:1451-1457, 2013.-   Goti et al., J. Neurochem., 76:498-508, 2001.-   Granholm et al., J. Pharmacol. Exp. Ther., 268:448-459, 1994.-   Hu et al., Cancer Res., 56, 3055-3061, 1996-   Huang and Liu, Annu. Rev. Biomed. Eng., 13:507-530, 2011.-   Iwai, et al., Chem. Pharm. Bull., 34:4724-4730, 1986-   Iwane, et al., Biochem. Biophys. Res. Comm., 171:116-122, 1990-   Juliano et al., Nucleic Acid Ther., 24:101-113, 2014.-   Kanaya, et al., Gene, 83:65-74, 1989-   Kim et al., Biomed. Chromatogr., 27:306-310, 2013.-   Kurakhmaeva et al., J. Drug Target., 17:564-574, 2009.-   Liu et al. Cell Mol. Biol., 49(2):209-216, 2003-   Mandel, Curr. Opin. Mol. Ther., 12:240-247, 2010.-   McArthur et al., Neurology, 54:1080-1088, 2000.-   McMahon et al., Expert Opin. Drug Deliv., 11:231-247, 2014.-   Miklan et at., Biopolymers., 92:489-501, 2009.-   Olivier, NeuroRx, 2:108-119, 2005.-   Pinto Reis et al., Nanomedicine 2:53-65, 2006.-   Provost et al., CR Biol. 326:841-51, 2003.-   Shahzad et al., Neoplasia., 13:309-319, 2011.-   Sofroniew et al., Annu. Rev. Neurosci., 24:1217-1281, 2001.-   Sowery et al., BJU Int., 102:389-397, 2008.-   Tria et al., Exp. Neurol., 127:178-183, 1994.-   Vaishya et al., Expert Opin. Drug Deliv., 12:415-440, 2015.-   Wahlberg et al., J. Neurosurg., 117:340-347, 2012.-   Wu et al., Clin. Cancer Res., 19:4740-4749, 2013.-   Xie et al., J. Control. Release, 105:106-119, 2005.-   Zhang et al., Biomaterials 33:4889-4906, 2012.-   Zhang et al., Pharm. Res., 26:1561-1580, 2009.

1. A composition comprising: (a) a therapeutic agent; (b) anα-tocopheryl compound; (c) a phospholipid composition; and (d) a steroidor steroid derivative, wherein the composition is formulated as ananoparticle and the α-tocopheryl compound is substantially located onthe surface of the nanoparticle.
 2. A composition comprising: (a) atherapeutic agent; (b) an α-tocopheryl compound; (c) a phospholipidcomposition; (d) a steroid or steroid derivative, and (e) anapolipoprotein; wherein the composition is formulated as a nanoparticleand the α-tocopheryl compound is substantially located on the surface ofthe nanoparticle.
 3. The composition of claim 1, wherein the therapeuticagent is a therapeutic protein.
 4. The composition of claim 3, whereinthe therapeutic protein is a growth factor, a neurotrophic factor, anantibody or mixture of antibodies, a protein that binds to VEGF and/orPIGF. 5-18. (canceled)
 19. The composition of claim 3, wherein thetherapeutic protein is a mixture of a therapeutic protein and apolycationic protein molecule. 20-21. (canceled)
 22. The composition ofclaim 1, wherein the therapeutic agent is a chemotherapeutic compound.23-24. (canceled)
 25. The composition of claim 1, wherein thetherapeutic agent is a therapeutic oligonucleotide. 26-29. (canceled)30. The composition of claim 1, wherein the therapeutic agent is acomposition comprising a chemotherapeutic agent and a therapeuticoligonucleotide. 31-33. (canceled)
 34. The composition according toclaim 1, wherein the α-tocopheryl compound is a pegylated derivative ofα-tocopheryl. 35-41. (canceled)
 42. The composition according to claim1, wherein the phospholipid composition comprises two or morephospholipids. 43-66. (canceled)
 67. The composition according to claim1, wherein the phospholipid composition further comprises a second orthird phospholipid. 68-93. (canceled)
 94. The composition according toclaim 1, further comprising an endosomal escaping agent.
 95. Thecomposition according to claim 1, wherein the steroid or steroidderivative is a cholesterol ester_((C≦24)).
 96. (canceled)
 97. Thecomposition according to claim 1, wherein composition further comprisesan apoliprotein. 98-99. (canceled)
 100. The composition according toclaim 1, wherein the composition further comprises a cell permeablizingagent. 101.-102. (canceled)
 103. The composition according to claim 1,wherein the composition further comprises a targeting agent. 104.(canceled)
 105. The composition according to claim 1, wherein the ratioof the phospholipid composition to the steroid or steroid derivative isfrom about 1:5 to about 15:1. 106-108. (canceled)
 109. The compositionaccording to claim 1, wherein the ratio of the phospholipids in thephospholipid composition comprises a phosphatidylcholine tosphingomyelin ratio from about 10:1 to about 1:2. 110-111. (canceled)112. The composition according to claim 1, wherein the ratio of thephospholipids in the phospholipid composition comprises aphosphatidylcholine to phospholtidylserine ratio from about 25:1 toabout 1:1. 113-114. (canceled)
 115. The composition according to claim1, wherein the steroid or steroid derivative comprises 0.5 w/w % toabout 12.5 w/w % of the composition. 116-117. (canceled)
 118. Thecomposition according to claim 1, wherein the phospholipid compositioncomprises from about 10 w/w % to about 45 w/w % of the composition.119-120. (canceled)
 121. The composition according to claim 1, whereinthe α-tocopheryl compound comprises from about 5 w/w % to about 60 w/w %of the composition. 122-123. (canceled)
 124. The composition accordingto claim 1, wherein the therapeutic agent comprises from about 0.5 w/w %to about 25 w/w %. 125-127. (canceled)
 128. The composition according toclaim 1, wherein the composition comprises the therapeutic agent and apolycationic molecule in a ratio from about 10:1 to about 1:10. 129-130.(canceled)
 131. The composition according to claim 1, wherein theapolipoprotein comprises from about 20 w/w % to about 70 w/w % of thecomposition. 132-133. (canceled)
 134. The composition according to claim1, wherein the nanoparticle further comprisesmonogalactosyldiacylglycerol.
 135. The composition according to claim 1,wherein the nanoparticle has a particle size from about 100 nm to about500 nm. 136-139. (canceled)
 140. The composition according to claim 1,wherein the polydispersity index is less than 0.3. 141-145. (canceled)146. A method of preparing a therapeutic agent-loaded nanoparticlecomprising: (a) admixing a composition with an organic solvent andcholesterol, a composition with an organic solvent and a phospholipidcomposition, a composition with an organic solvent and an α-tocopherylcompound, and a composition with a solvent and a therapeutic agent toform a first reaction mixture; (b) removing the organic solvent from thefirst reaction mixture to form a second reaction mixture; (c) admixingthe second reaction mixture to water by using a homogenizer or asonication probe to form a prototype nanoparticle; and (d) admixing oneor more therapeutic agents with the prototype nanoparticle to form atherapeutic agent-loaded nanoparticle.
 147. A method of preparing atherapeutic agent-loaded HDL mimicking nanoparticle comprising: (a)admixing a composition with an organic solvent and cholesterol, acomposition with an organic solvent and a phospholipid composition, anda composition with an organic solvent and an α-tocopheryl compound toform a first reaction mixture; (b) removing the organic solvent from thefirst reaction mixture to form a second reaction mixture; (c) admixingthe second reaction mixture to water to form a prototype nanoparticle.(d) admixing one or more therapeutic agents with the prototypenanoparticle to form a therapeutic agent-loaded nanoparticle; and (e)admixing the therapeutic agent-loaded nanoparticle with apolipoproteinA-I to form a therapeutic agent-loaded HDL-mimicking nanoparticle.148-180. (canceled)
 181. A composition prepared according to the methodsof any one of method of claim
 146. 182. A method of treating a diseaseor disorder in a patient comprising administering to the patient atherapeutically effective amount of a composition according to claim 1.183-203. (canceled)
 204. A method of inducing neuronal growth comprisingadministering a composition according to claim
 1. 205.-207. (canceled)